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Sample records for neuron-glial cell cocultures

  1. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...

  2. Role of IFN-gamma and LPS on Neuron/Glial Co-Cultures Infected by Neospora caninum

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    Erica Etelvina Viana De Jesus

    2014-10-01

    Full Text Available Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 μg/mL of LPS on infected rat neuron/glial co-cultures. After 72 hours of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not, meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001, this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%. The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in CNS

  3. Enzymatic removal of polysialic acid from neural cell adhesion molecule interrupts gonadotropin releasing hormone (GnRH) neuron-glial remodeling.

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    Kumar, Sushil; Parkash, Jyoti; Kataria, Hardeep; Kaur, Gurcharan

    2012-01-02

    There is abundant evidence to prove that the astrocytes are highly dynamic cell type in CNS and under physiological conditions such as reproduction, these cells display a remarkable structural plasticity especially at the level of their distal processes ensheathing the gonadotropin releasing hormone (GnRH) axon terminals. The morphology of GnRH axon terminals and astrocytes in the median eminence region of hypothalamus show activity dependent structural plasticity during different phases of estrous cycle. In the current study, we have assessed the functional contribution of ∞-2,8-linked polysialic acid (PSA) on neural cell adhesion molecule (PSA-NCAM) in this neuronal-glial plasticity using both in vitro and in vivo model systems. In vivo experiments were carried out after stereotaxic injection of endoneuraminidase enzyme (endo-N) near median eminence region of hypothalamus to specifically remove PSA residues on NCAM followed by localization of GnRH, PSA-NCAM and glial fibrillary acidic protein (GFAP) by immunostaining. Using in vitro model, structural remodeling of GnV-3 cells, (a conditionally immortalized GnRH cell line) co-cultured with primary astrocytes was studied after treating the cells with endo-N. Marked morphological changes were observed in GnRH axon terminals in proestrous phase rats and control GnV-3 cells as compared to endo-N treatment i.e. after removal of PSA. The specificity of endo-N treatment was also confirmed by studying the expression of PSA-NCAM by Western blotting in cultures treated with and without endo-N. Removal of PSA from surfaces with endo-N prevented stimulation associated remodeling of GnRH axon terminals as well as their associated glial cells under both in vivo and in vitro conditions. The current data confirms the permissive role of PSA to promote dynamic remodeling of GnRH axon terminals and their associated glia during reproductive cycle in rats. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex.

    Directory of Open Access Journals (Sweden)

    María García-Amado

    Full Text Available Cell number alterations in the amygdaloid complex (AC might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL, corticomedial and central groups, 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3 and mean cell numbers (x10(6 were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.

  5. Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex.

    Science.gov (United States)

    García-Amado, María; Prensa, Lucía

    2012-01-01

    Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3) and mean cell numbers (x10(6)) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.

  6. Chemokines in neuron-glial cell interaction and pathogenesis of neuropathic pain.

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    Zhang, Zhi-Jun; Jiang, Bao-Chun; Gao, Yong-Jing

    2017-09-01

    Neuropathic pain resulting from damage or dysfunction of the nervous system is a highly debilitating chronic pain state and is often resistant to currently available treatments. It has become clear that neuroinflammation, mainly mediated by proinflammatory cytokines and chemokines, plays an important role in the establishment and maintenance of neuropathic pain. Chemokines were originally identified as regulators of peripheral immune cell trafficking and were also expressed in neurons and glial cells in the central nervous system. In recent years, accumulating studies have revealed the expression, distribution and function of chemokines in the spinal cord under chronic pain conditions. In this review, we provide evidence showing that several chemokines are upregulated after peripheral nerve injury and contribute to the pathogenesis of neuropathic pain via different forms of neuron-glia interaction in the spinal cord. First, chemokine CX3CL1 is expressed in primary afferents and spinal neurons and induces microglial activation via its microglial receptor CX3CR1 (neuron-to-microglia signaling). Second, CCL2 and CXCL1 are expressed in spinal astrocytes and act on CCR2 and CXCR2 in spinal neurons to increase excitatory synaptic transmission (astrocyte-to-neuron signaling). Third, we recently identified that CXCL13 is highly upregulated in spinal neurons after spinal nerve ligation and induces spinal astrocyte activation via receptor CXCR5 (neuron-to-astrocyte signaling). Strategies that target chemokine-mediated neuron-glia interactions may lead to novel therapies for the treatment of neuropathic pain.

  7. Diverse Physiological Roles of Calcitonin Gene-Related Peptide in Migraine Pathology: Modulation of Neuronal-Glial-Immune Cells to Promote Peripheral and Central Sensitization.

    Science.gov (United States)

    Durham, Paul L

    2016-08-01

    The neuropeptide calcitonin gene-related peptide (CGRP) is implicated in the underlying pathology of migraine by promoting the development of a sensitized state of primary and secondary nociceptive neurons. The ability of CGRP to initiate and maintain peripheral and central sensitization is mediated by modulation of neuronal, glial, and immune cells in the trigeminal nociceptive signaling pathway. There is accumulating evidence to support a key role of CGRP in promoting cross excitation within the trigeminal ganglion that may help to explain the high co-morbidity of migraine with rhinosinusitis and temporomandibular joint disorder. In addition, there is emerging evidence that CGRP facilitates and sustains a hyperresponsive neuronal state in migraineurs mediated by reported risk factors such as stress and anxiety. In this review, the significant role of CGRP as a modulator of the trigeminal system will be discussed to provide a better understanding of the underlying pathology associated with the migraine phenotype.

  8. Neuronal-glial trafficking

    International Nuclear Information System (INIS)

    Bachelard, H.S.

    2001-01-01

    Full text: The name 'glia' originates from the Greek word for glue, because astro glia (or astrocytes) were thought only to provide an anatomical framework for the electrically-excitable neurones. However, awareness that astrocytes perform vital roles in protecting the neurones, which they surround, emerged from evidence that they act as neuroprotective K + -sinks, and that they remove potentially toxic extracellular glutamate from the vicinity of the neurones. The astrocytes convert the glutamate to non-toxic glutamine which is returned to the neurones and used to replenish transmitter glutamate. This 'glutamate-glutamine cycle' (established in the 1960s by Berl and his colleagues) also contributes to protecting the neurones against a build-up of toxic ammonia. Glial cells also supply the neurones with components for free-radical scavenging glutathione. Recent studies have revealed that glial cells play a more positive interactive role in furnishing the neurones with fuels. Studies using radioactive 14 C, 13 C-MRS and 15 N-GCMS have revealed that glia produce alanine, lactate and proline for consumption by neurones, with increased formation of neurotransmitter glutamate. On neuronal activation the release of NH 4 + and glutamate from the neurones stimulates glucose uptake and glycolysis in the glia to produce more alanine, which can be regarded as an 'alanine-glutamate cycle' Use of 14 C-labelled precursors provided early evidence that neurotransmitter GABA may be partly derived from glial glutamine, and this has been confirmed recently in vivo by MRS isotopomer analysis of the GABA and glutamine labelled from 13 C-acetate. Relative rates of intermediary metabolism in glia and neurones can be calculated using a combination of [1- 13 C] glucose and [1,2- 13 C] acetate. When glutamate is released by neurones there is a net neuronal loss of TCA intermediates which have to be replenished. Part of this is derived from carboxylation of pyruvate, (pyruvate carboxylase

  9. Cell-surface expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) in heterogeneous cultures of marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Russell, Katie C; Tucker, H Alan; Bunnell, Bruce A; Andreeff, Michael; Schober, Wendy; Gaynor, Andrew S; Strickler, Karen L; Lin, Shuwen; Lacey, Michelle R; O'Connor, Kim C

    2013-10-01

    Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. Sc(LO)NG2(HI) and Sc(LO)NG2(HI)CD146(HI) MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls. The Sc(LO) gate enriches for rapidly dividing cells. Addition of the NG2(HI) gate increases cell survival to 1.5 times the parental control. Further addition of the CD146(HI) gate does not significantly

  10. Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

    Science.gov (United States)

    Pan, Wei-Kang; Yu, Hui; Wu, A-Li; Gao, Ya; Zheng, Bai-Jun; Li, Peng; Yang, Wei-Li; Huang, Qiang; Wang, Huai-Jie; Ge, Xin

    2016-08-03

    Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both Pcells to differentiate into neuronal cells than that of EGF (Pglial differentiation compared with addition of bFGF (Pcells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

  11. The digestive neuronal-glial-epithelial unit: a new actor in gut health and disease.

    Science.gov (United States)

    Neunlist, Michel; Van Landeghem, Laurianne; Mahé, Maxime M; Derkinderen, Pascal; des Varannes, Stanislas Bruley; Rolli-Derkinderen, Malvyne

    2013-02-01

    The monolayer of columnar epithelial cells lining the gastrointestinal tract--the intestinal epithelial barrier (IEB)--is the largest exchange surface between the body and the external environment. The permeability of the IEB has a central role in the regulation of fluid and nutrient intake as well as in the control of the passage of pathogens. The functions of the IEB are highly regulated by luminal as well as internal components, such as bacteria or immune cells, respectively. Evidence indicates that two cell types of the enteric nervous system (ENS), namely enteric neurons and enteric glial cells, are potent modulators of IEB functions, giving rise to the novel concept of a digestive 'neuronal-glial-epithelial unit' akin to the neuronal-glial-endothelial unit in the brain. In this Review, we summarize findings demonstrating that the ENS is a key regulator of IEB function and is actively involved in pathologies associated with altered barrier function.

  12. Energy metabolism in neuronal/glial induction and in iPSC models of brain disorders.

    Science.gov (United States)

    Mlody, Barbara; Lorenz, Carmen; Inak, Gizem; Prigione, Alessandro

    2016-04-01

    The metabolic switch associated with the reprogramming of somatic cells to pluripotency has received increasing attention in recent years. However, the impact of mitochondrial and metabolic modulation on stem cell differentiation into neuronal/glial cells and related brain disease modeling still remains to be fully addressed. Here, we seek to focus on this aspect by first addressing brain energy metabolism and its inter-cellular metabolic compartmentalization. We then review the findings related to the mitochondrial and metabolic reconfiguration occurring upon neuronal/glial specification from pluripotent stem cells (PSCs). Finally, we provide an update of the PSC-based models of mitochondria-related brain disorders and discuss the challenges and opportunities that may exist on the road to develop a new era of brain disease modeling and therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Plasticity of Neuron-Glial Transmission: Equipping Glia for Long-Term Integration of Network Activity

    Directory of Open Access Journals (Sweden)

    Wayne Croft

    2015-01-01

    Full Text Available The capacity of synaptic networks to express activity-dependent changes in strength and connectivity is essential for learning and memory processes. In recent years, glial cells (most notably astrocytes have been recognized as active participants in the modulation of synaptic transmission and synaptic plasticity, implicating these electrically nonexcitable cells in information processing in the brain. While the concept of bidirectional communication between neurons and glia and the mechanisms by which gliotransmission can modulate neuronal function are well established, less attention has been focussed on the computational potential of neuron-glial transmission itself. In particular, whether neuron-glial transmission is itself subject to activity-dependent plasticity and what the computational properties of such plasticity might be has not been explored in detail. In this review, we summarize current examples of plasticity in neuron-glial transmission, in many brain regions and neurotransmitter pathways. We argue that induction of glial plasticity typically requires repetitive neuronal firing over long time periods (minutes-hours rather than the short-lived, stereotyped trigger typical of canonical long-term potentiation. We speculate that this equips glia with a mechanism for monitoring average firing rates in the synaptic network, which is suited to the longer term roles proposed for astrocytes in neurophysiology.

  14. [Research progress of cell co-culture method].

    Science.gov (United States)

    Qin, Yanqin; Chen, Yulong; Li, Jiansheng

    2016-08-01

    Cell culture technology is the most commonly used method in the in vitro experiments at present. However, monolayer cell culture technology has been unable to meet the demand of the researchers. This is because that monolayer cell culture cannot mimic the cellular environment in which multiple cells interact with each other in the body. We cannot discuss the relationship of many cells, because we do not know the relationship between cells through a single kind of cell. So cell co-culture medicine arises at the historic moment for the demand. With the development of research method in recent years, cell co-culture method also has been improved in practice: from direct contact co-cultures to indirect contact co-cultures, from two-dimensional co-cultures to three-dimensional co-cultures. Cell co-culture method is closer to the human body. It is also more advantageous to study the interaction among cells. Nowadays, there are more researchers tend to select this method to study the physiological and pathological in vitro model, tissue engineering, and cell differentiation research. At the same time, it has become the focus of drug research and development, drug analysis, mechanism of drug action, and drug targets. This article will review the studies of cell co-culture method, summarize advantages and disadvantages of various methods, so as to promote improvement of cell culture methods, to build cells co-culture system that more close to human body, and build the in vitro model that simulate internal circulation of human body further.

  15. [Application of cell co-culture techniques in medical studies].

    Science.gov (United States)

    Luo, Yun; Sun, Gui-Bo; Qin, Meng; Yao, Fan; Sun, Xiao-Bo

    2012-11-01

    As the cell co-culture techniques can better imitate an in vivo environment, it is helpful in observing the interactions among cells and between cells and the culture environment, exploring the effect mechanisms of drugs and their possible targets and filling the gaps between the mono-layer cell culture and the whole animal experiments. In recently years, they has attracted much more attention from the medical sector, and thus becoming one of research hotspots in drug research and development and bio-pharmaceutical fields. The cell co-culture techniques, including direct and indirect methods, are mainly used for studying pathological basis, new-type treatment methods and drug activity screening. Existing cell co-culture techniques are used for more pharmacological studies on single drug and less studies on interaction of combined drugs, such as collaborative compatibility and attenuation and synergistic effect among traditional Chinese medicines (TCMs). In line with the action characteristics of multi-component and multi-target, the cell co-culture techniques provide certain reference value for future studies on the effect and mechanism of combined TCMs on organisms as well as new methods for studies on TCMs and their compounds. This essay summarizes cell co-culture methods and their application and look into the future of their application in studies on TCMs and compounds.

  16. Lack of expression of the chondroitin sulphate proteoglycan neuron-glial antigen 2 on candidate stem cell populations in paediatric acute myeloid leukaemia/abn(11q23) and acute lymphoblastic leukaemia/t(4;11)

    NARCIS (Netherlands)

    J. Neudenberger (J.); M. Hotfilder (Marc); A. Rosemann (Annegret); C. Langebrake (C.); D. Reinhardt (Dirk); R. Pieters (Rob); A. Schrauder (André); M. Schrappe (Martin); S. Röttgers (Silja); J. Harbott (Jochen); J. Vormoor (Josef)

    2006-01-01

    textabstractIt has increasingly been acknowledged that only a few leukaemic cells possess the capability to renew themselves and that only these self-renewing leukaemic stem cells are able to initiate relapses. Therefore, these leukaemic stem cells should be the target cells for therapy and for

  17. Mixed neuronal-glial tumor in the temporal lobe of an infant: a case report.

    Science.gov (United States)

    Yano, Hirohito; Saigoh, Chiemi; Nakayama, Noriyuki; Hirose, Yoshinobu; Abe, Masato; Ohe, Naoyuki; Ozeki, Michio; Shinoda, Jun; Iwama, Toru

    2013-10-02

    Tumors that arise in the temporal lobes of infants and spread to the neural system are limited to several diagnoses. Herein, we present an infantile case of a temporal tumor showing neuronal and glial differentiation. The patient was a 9-month-old boy with low body weight due to intrauterine growth retardation. At 9 months after birth, he presented partial seizures. Computed tomography scanning revealed a mass (35 * 40 mm) in the left temporal lobe. Isointensity was noted on magnetic resonance T1-weighted images and fluid attenuation inversion recovery images. The tumor was heterogeneously enhanced with gadolinium. Positron emission tomography showed high methionine uptake in the tumor. During surgery, the tumor, which was elastic and soft and bled easily, was gross totally resected. A moderately clear boundary was noted between the tumor and normal brain parenchyma. Histologically, the tumor mainly comprised a ganglioglioma-like portion and short spindle cells at different densities. The former was immunohistochemically positive for some kinds of neuronal markers including synaptophysin. The spindle cells were positive for glial fibrillary acidic protein, but desmoplasia was not observed. The tumor contained both neuronal and glial elements; the former were the main constituents of the tumor and included several ganglion-like cells. Because neuronal elements gradually transited to glial cells, a mixed neuronal-glial tumor was diagnosed. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2045126100982604.

  18. Bone regeneration using coculture of mesenchymal stem cells and angiogenic cells

    Science.gov (United States)

    Ma, Jin-Ling; van den Beucken, Jeroen J. J. P.; Pan, Ju-Li; Cui, Fu-Zhai; Chen, Su

    2014-03-01

    Cellular strategies remain a crucial component in bone tissue engineering (BTE). So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the tissue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding (i) critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and (ii) the usage of monocultures in clinical application in BTE.

  19. Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

    Science.gov (United States)

    Yamazoe, Hironori; Ichikawa, Takashi; Hagihara, Yoshihisa; Iwasaki, Yasuhiko

    2016-02-01

    Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a

  20. Neuron-Glial Interactions in Blood-Brain Barrier Formation

    OpenAIRE

    Banerjee, Swati; Bhat, Manzoor A.

    2007-01-01

    The blood brain barrier (BBB) evolved to preserve the microenvironment of the highly excitable neuronal cells to allow for action potential generation and propagation. Intricate molecular interactions between two main cell types, the neurons and the glial cells, form the underlying basis of the critical functioning of the nervous system across species. In invertebrates, interactions between neurons and glial cells are central in establishing a functional BBB. However, in vertebrates, the BBB ...

  1. Triple co-culture cell model as an in vitro model for oral particulate vaccine systems

    DEFF Research Database (Denmark)

    Nielsen, Line Hagner; De Rossi, C.; Lehr, C-M.

    values of the co-cultures were found to be 860-1340 Ω∙cm2; the formulations were incubated with the co-cultures at this time point. From confocal microscopy images, it was observed that the THP-1 cells (macrophages) migrated into the overlying Caco-2 cell monolayer when the co-cultures were incubated......A triple co-culture cell model of Caco-2 cells, dendritic cells and macrophages (Figure 1) has previously been developed for studying intestinal permeability in a state of inflammation [1],[2]. The aim of this study was to investigate the applicability of this cell model for testing...... the model antigen ovalbumin was spray dried to obtain a particulate vaccine model system for testing in the cell model. The precursors were shown to form cubosomes when dispersed in aqueous medium, and was therefore used as the vaccine formulation for testing on the co-cultures. After 11 days, the TEER...

  2. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    International Nuclear Information System (INIS)

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-01

    Research highlights: → The proliferation of dramatic increased by co-cultured with Sertoli cells. → VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. → The MHC expression of ECs induced by INF-γ and IL-6, IL-8 and sICAM induced by TNF-α decreased respectively after co-cultured with Sertoli cells. → ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10 3 , 1 x 10 4 or 1 x 10 5 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10 4 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P 4 cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single

  3. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli

  4. Neural differentiation of adipose-derived stem cells by indirect co-culture with Schwann cells

    Directory of Open Access Journals (Sweden)

    Li Xiaojie

    2009-01-01

    Full Text Available To investigate whether adipose-derived stem cells (ADSCs could be subject to neural differentiation induced only by Schwann cell (SC factors, we co-cultured ADSCs and SCs in transwell culture dishes. Immunoassaying, Western blot analysis, and RT-PCR were performed (1, 3, 7, 14 d and the co-cultured ADSCs showed gene and protein expression of S-100, Nestin, and GFAP. Further, qRT-PCR disclosed relative quantitative differences in the above three gene expressions. We think ADSCs can undergo induced neural differentiation by being co-cultured with SCs, and such differentia­tions begin 1 day after co-culture, become apparent after 7 days, and thereafter remain stable till the 14th day.

  5. Boron Nitride Nanotube-Mediated Stimulation of Cell Co-Culture on Micro-Engineered Hydrogels

    OpenAIRE

    Ricotti, L.; Fujie, T.; Vazão, H.; Ciofani, G.; Marotta, R.; Brescia, R.; Filippeschi, C.; Corradini, I.; Matteoli, M.; Mattoli, V.; Ferreira, L.; Menciassi, A.

    2013-01-01

    In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric ...

  6. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

    Directory of Open Access Journals (Sweden)

    Xingfu Li

    2016-01-01

    Full Text Available Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2 and decreased type I collagen (COL1 protein expression levels. SRY-box 9 (SOX9 mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

  7. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes.

    Science.gov (United States)

    Li, Xingfu; Duan, Li; Liang, Yujie; Zhu, Weimin; Xiong, Jianyi; Wang, Daping

    2016-01-01

    Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

  8. Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells.

    Science.gov (United States)

    Ishii, Tomohiro; Kawakami, Emiko; Endo, Kentaro; Misawa, Hidemi; Watabe, Kazuhiko

    2017-10-01

    Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration. © 2017 Japanese Society of Neuropathology.

  9. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.

    Science.gov (United States)

    Kaji, Hirokazu; Camci-Unal, Gulden; Langer, Robert; Khademhosseini, Ali

    2011-03-01

    Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell-cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell-cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. 2010 Elsevier B.V. All rights reserved.

  10. [Biological characteristics of mesenchymal stem cell and hematopoietic stem cell in the co-culture system].

    Science.gov (United States)

    Wei, Wei; Xu, Chao; Ye, Zhi-Yong; Huang, Xiao-Jun; Yuan, Jia-En; Ma, Tian-Bao; Lin, Han-Biao; Chen, Xiu-Qiong

    2016-10-25

    The aim of the present study was to obtain the qualified hematopoietic stem/progenitor cells (HSC/HPC) and human umbilical cord-mesenchymal stem cells (MSC) in vitro in the co-culture system. Cord blood mononuclear cells were separated from umbilical cord blood by Ficoll lymphocyte separation medium, and then CD34 + HSC was collected by MACS immunomagnetic beads. The selected CD34 + HSC/HPC and MSC were transferred into culture flask. IMDM culture medium with 15% AB-type cord plasma supplemented with interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt-3L) factors were used as the co-culture system for the amplification of HSC/HPC and MSC. The cellular growth status and proliferation on day 6 and 10 after co-culture were observed by using inverted microscope. The percentage of positive expression of CD34 in HSC/HPC, as well as the percentages of positive expressions of CD105, CD90, CD73, CD45, CD34 and HLA-DR in the 4 th generation MSC, was tested by flow cytometry. Semisolid colony culture was used to test the HSC/HPC colony forming ability. The osteogenic, chondrogenesis and adipogenic ability of the 4 th generation MSC were assessed. The karyotype analysis of MSC was conducted by colchicines. The results demonstrated that the HSC/HPC of co-culture group showed higher ability of amplification, CFU-GM and higher CD34 + percentage compared with the control group. The co-cultured MSC maintained the ability to differentiate into bone cells, fat cells and chondrocytes. And the karyotype stability of MSC remained normal. These results reveal that the appropriate co-culture system for MSC and HSC is developed, and via this co-culture system we could gain both two kinds of these cells. The MSCs under the co-culture system maintain the biological characteristics. The CFU-GM ability, cell counting and the flow cytometry results of HSC/HPC under the co-culture system are conform to the criterion, showing that

  11. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People' s Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  12. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    International Nuclear Information System (INIS)

    Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

    2012-01-01

    Highlights: ► Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). ► Presence of SCs dramatically increased proliferation and migration of UCMSCs. ► Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of “nurse” cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  13. Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization.

    Science.gov (United States)

    Shah, Amita R; Wenke, Joseph C; Agrawal, Chandra Mauli

    2016-01-01

    Tissue-engineering scaffolds are often seeded with a single type of cell, but there has been more focus on cocultures to improve angiogenesis and bone formation for craniofacial applications. Investigation of bone-derived osteoblasts (OBs) is important because of the use of bone grafts and migration of OBs from native bone into constructs in vivo and therefore, their contribution to bone formation in vivo. The limitation of primary OBs has been their inability to mineralize without osteogenic factors in vitro. Through coculture of OBs and endothelial cells (ECs) and manipulation of the coculture ratio, mineralization can be achieved without osteogenic media or additional growth factors, thus enhancing their utility for tissue-engineering applications. An optimal ratio of EC/OB for vasculogenesis and mineralization has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios, namely, 10:1, 5:1, 1:1, 1:5, and 1:10 with EC and OB monocultures as controls. The number of vasculogenic networks in a collagen matrix was highest in ratios of 5:1 and 1:1. ECs lined up and formed capillary-like networks by day 10, which was not seen in the other groups. On polystyrene, cells were cocultured with ECs and OBs in direct contact (direct coculture) or separated by a transwell membrane (indirect coculture). At day 21, Alizarin Red staining showed mineralization on the 1:5 and 1:10 direct coculture ratios, with 1:5 having more mineralization nodules present than 1:10. No mineralization was seen in other direct coculture ratios or in any of the indirect coculture ratios. Alkaline phosphatase secretion was highest in the 1:5 direct coculture group. Vascular endothelial growth factor secretion from OBs was present in the 1:5 and 1:10 direct coculture ratios at all time points and inhibited after day 1 in other coculture groups. To improve vasculogenesis, cocultures of primary human ECs and OBs in ratios

  14. Advances in tissue engineering through stem cell-based co-culture.

    Science.gov (United States)

    Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-05-01

    Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

  15. Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

    Science.gov (United States)

    Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

    2014-05-01

    Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Prefrontal changes in the glutamate-glutamine cycle and neuronal/glial glutamate transporters in depression with and without suicide

    NARCIS (Netherlands)

    Zhao, J.; Verwer, R.W.H.; van Wamelen, D.J.; Qi, X.R.; Gao, S.F.; Lucassen, P.J.; Swaab, D.F.

    2016-01-01

    There are indications for changes in glutamate metabolism in relation to depression or suicide. The glutamate-glutamine cycle and neuronal/glial glutamate transporters mediate the uptake of the glutamate and glutamine. The expression of various components of the glutamate-glutamine cycle and the

  17. Visualization of the melanosome transfer-inhibition in a mouse epidermal cell co-culture model.

    Science.gov (United States)

    Kim, Hae Jong; Kazi, Julhash U; Lee, You-Ree; Nguyen, Dung H; Lee, Hyang-Bok; Shin, Jeong-Hyun; Soh, Jae-Won; Kim, Eun-Ki

    2010-02-01

    Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

  18. Human endothelial and foetal femur-derived stem cell co-cultures modulate osteogenesis and angiogenesis.

    Science.gov (United States)

    Inglis, Stefanie; Christensen, David; Wilson, David I; Kanczler, Janos M; Oreffo, Richard O C

    2016-01-18

    A dynamic vasculature is a prerequisite for bone formation where the interaction of bone cells and endothelial cells is essential for both the development and the healing process of bone. Enhanced understanding of the specific mediators involved in bone cell and endothelial cell interactions offers new avenues for skeletal regenerative applications. This study has investigated the osteogenic and angiogenic potential of co-cultures of human foetal diaphyseal or epiphyseal cells with human umbilical vein endothelial cells (HUVEC) in the presence and absence of vascular endothelial growth factor (VEGF) supplementation. Early osteogenic activities of the co-cultures (± VEGF) were assessed by alkaline phosphatase (ALP) activity. Osteogenic and angiogenic gene expression was measured using quantitative polymerase chain reaction. An ex vivo organotypic embryonic chick (E11) femur culture model was used to determine the osteogenic effects of VEGF as determined using micro-computed tomography (μCT) and Alcian blue/Sirius red histochemistry and immunocytochemistry for expression of CD31. ALP activity and gene expression of ALP and Type-1 collagen was enhanced in foetal skeletal/HUVECs co-cultures. In foetal diaphyseal/HUVECs co-cultures, VEGF reduced the levels of ALP activity and displayed a negligible effect on von Willebrand factor (vWF) and VEGF gene expression. In contrast, VEGF supplementation was observed to significantly increase FLT-1 and KDR gene expression in co-cultures with modulation of expression enhanced, compared to VEGF skeletal monocultures. In the organotypic chick model, addition of VEGF significantly enhanced bone formation, which coincided with elevated levels of CD31-positive cells in the mid-diaphyseal region of the femurs. These studies demonstrate a differential skeletal response of early foetal skeletal cells, when co-cultured with endothelial cells and the potential of co-culture models for bone repair. The differential effect of VEGF

  19. Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system.

    Science.gov (United States)

    Varshney, Rohan R; Zhou, Ruijie; Hao, Jinghua; Yeo, Suan Siong; Chooi, Wai Hon; Fan, Jiabing; Wang, Dong-An

    2010-09-01

    A co-culture strategy has been developed in this study wherein rabbit synovial mesenchymal stem cells (SMSCs) are co-cultured with growth factor (GF) transfected articular chondrocytes. Toward this end, both SMSCs and early passage rabbit articular chondrocytes that had been adenovirally transduced with transforming growth factor-beta 3 (TGF-beta3) gene were separately encapsulated in alginate beads and co-cultured in the same pool of chondrogenic medium. The chondrocytes act as transfected companion cells (TCCs) providing GF supply to induce chondrogenic differentiation of SMSCs that play the role of therapeutic progenitor cells (TPCs). Against the same TCC based TGF-beta3 release profile, the co-culture was started at different time points (Day 0, Day 10 and Day 20) but made to last for identical periods of exposure (30 days) so that the exposure conditions could be optimized in terms of initiation and duration. Transfection of TCCs prevents the stem cell based TPCs from undergoing the invasive procedure. It also prevents unpredictable complications in the TPCs caused by long-term constitutive over-expression of a GF. The adenovirally transfected TCCs exhibit a transient GF expression which results in a timely termination of GF supply to the TPCs. The TCC-sourced transgenic TGF-beta3 successfully induced chondrogenesis in the TPCs. Real-time PCR results show enhanced expression of cartilage markers and immuno/histochemical staining for Glycosaminoglycans (GAG) and Collagen II also shows abundant extracellular matrix (ECM) production and chondrogenic morphogenesis in the co-cultured TPCs. These results confirm the efficacy of directing stem cell differentiation towards chondrogenesis and cartilage tissue formation by co-culturing them with GF transfected chondrocytes.

  20. Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes

    Directory of Open Access Journals (Sweden)

    Jie Shi

    2017-01-01

    Full Text Available Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1 adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2 the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes.

  1. Campylobacter jejuni cocultured with epithelial cells reduces surface capsular polysaccharide expression.

    LENUS (Irish Health Repository)

    Corcionivoschi, N

    2012-02-01

    The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.

  2. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  3. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts

    Directory of Open Access Journals (Sweden)

    Matthew M. Cook

    2013-09-01

    Full Text Available Haematopoietic stem cell (HSC transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs or osteoblasts. Sorted Lineage− Sca-1+ c-kit+ (LSK haematopoietic stem/progenitor cells (HPC demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

  4. Boron nitride nanotube-mediated stimulation of cell co-culture on micro-engineered hydrogels.

    Directory of Open Access Journals (Sweden)

    Leonardo Ricotti

    Full Text Available In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa and a small thickness (∼ 12 µm. We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin. We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

  5. Boron nitride nanotube-mediated stimulation of cell co-culture on micro-engineered hydrogels.

    Science.gov (United States)

    Ricotti, Leonardo; Fujie, Toshinori; Vazão, Helena; Ciofani, Gianni; Marotta, Roberto; Brescia, Rosaria; Filippeschi, Carlo; Corradini, Irene; Matteoli, Michela; Mattoli, Virgilio; Ferreira, Lino; Menciassi, Arianna

    2013-01-01

    In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

  6. Quantifying co-cultured cell phenotypes in high-throughput using pixel-based classification.

    Science.gov (United States)

    Logan, David J; Shan, Jing; Bhatia, Sangeeta N; Carpenter, Anne E

    2016-03-01

    Biologists increasingly use co-culture systems in which two or more cell types are grown in cell culture together in order to better model cells' native microenvironments. Co-cultures are often required for cell survival or proliferation, or to maintain physiological functioning in vitro. Having two cell types co-exist in culture, however, poses several challenges, including difficulties distinguishing the two populations during analysis using automated image analysis algorithms. We previously analyzed co-cultured primary human hepatocytes and mouse fibroblasts in a high-throughput image-based chemical screen, using a combination of segmentation, measurement, and subsequent machine learning to score each cell as hepatocyte or fibroblast. While this approach was successful in counting hepatocytes for primary screening, segmentation of the fibroblast nuclei was less accurate. Here, we present an improved approach that more accurately identifies both cell types. Pixel-based machine learning (using the software ilastik) is used to seed segmentation of each cell type individually (using the software CellProfiler). This streamlined and accurate workflow can be carried out using freely available and open source software. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions.

    Science.gov (United States)

    Rao, Nikhil; Grover, Gregory N; Vincent, Ludovic G; Evans, Samantha C; Choi, Yu Suk; Spencer, Katrina H; Hui, Elliot E; Engler, Adam J; Christman, Karen L

    2013-11-01

    Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.

  8. Calcitriol enhances fat synthesis factors and calpain activity in co-cultured cells.

    Science.gov (United States)

    Choi, Hyuck; Myung, Kyuho

    2014-08-01

    We have conducted an in vitro experiment to determine whether calcitriol can act as a fat synthesizer and/or meat tenderizer when skeletal muscle cells, adipose tissue, and macrophages are co-cultured. When co-cultured, pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) expression increased, whereas decreased anti-inflammatory cytokine (IL-10 and IL-15) expression decreased in both C2C12 and 3T3-L1 cells. Calcitriol increased reactive oxygen species (ROS) production in the media. While adiponectin gene expression decreased, leptin, resistin, CCAAT-enhancer-binding protein-beta (C/EBP-β), and peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression was significantly (P cultured with two different cell types. Inducible nitric oxide synthase (iNOS) protein levels were also stimulated in the C2C12 and 3T3-L1 cells, but arginase l was attenuated by calcitriol. Cacitriol highly amplified (P = 0.008) µ-calpain gene expression in co-cultured C2C12 cells. The results showed an overall increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines of C2C12 and 3T3-L1 cells with calcitriol in co-culture systems. µ-Calpain protein was also augmented in differentiated C2C12 cells with calcitriol. These findings suggest that calcitriol can be used as not only fat synthesizer, but meat tenderizer, in meat-producing animals. © 2014 International Federation for Cell Biology.

  9. Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis.

    Science.gov (United States)

    Grässer, Ute; Bubel, Monika; Sossong, Daniela; Oberringer, Martin; Pohlemann, Tim; Metzger, Wolfgang

    2018-03-01

    Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy ® -technology and the cellular composition was analyzed by flow cytometry. Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase ® , whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size. Copyright © 2017 Elsevier GmbH. All rights reserved.

  10. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

    Science.gov (United States)

    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  11. Novel cell separation method for molecular analysis of neuron-astrocyte cocultures

    Directory of Open Access Journals (Sweden)

    Andrea eGoudriaan

    2014-01-01

    Full Text Available Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedite technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova et al., 1997, and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

  12. [Design and fabrication of a microfluidic chip for the co-culture of three cell types].

    Science.gov (United States)

    Wang, Shuaichao; Ge, Yuqing; Wu, Lei; Guo, Hui; Yang, Shiping; Jin, Qinghui

    2017-02-25

    Here a microfluidic chip with 'micro-dam' and 'micro-gap' has been designed and fabricated. It could isolate different cells and flow of medium in each region. It was found that the chip could realize the cells co-culture and patterning of human lung adenocarcinoma cell (A549), human embryonic lung fibroblast (HLF-1) and human endothelial cells (HUVECs), respectively. After 72 hours of culture, three kinds of cells grew well. It provided a developing technical platform for cell related research.

  13. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-01-01

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

  14. The proliferation and differentiation characteristics of co-cultured porcine preadipocytes and muscle satellite cells in vitro.

    Science.gov (United States)

    Yan, Jun; Gan, Lu; Yang, Haili; Sun, Chao

    2013-04-01

    To explore the proliferation and differentiation characteristics of co-cultured porcine preadipocytes and muscle satellite cells, preadipocytes and muscle satellite cells were isolated from the healthy nascent landrace. Oil Red O stain and desmin immunohistochemistry were used to identify the two solo-cultured cells. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to detect the proliferation characteristic of co-cultured cells, and the expression level of differentiation marker genes lipoprotein lipase (LPL), peroxisome proliferator-activated receptors (PPARγ), myogenic factor 5 (Myf5), myogenin (MyoG) were analyzed with reverse transcription PCR (RT-PCR) and western blot. The success of co-culture system was proved. In the co-cultured cells, slight lipid droplets were observed and appeared more slowly. The polykaryocytes fused into myotubes in co-cultured cells were less and relatively slow than that in solo myocytes. After fusion, the proliferation rate of co-cultured cells was higher than that in the solo-cultured muscle satellite cells (P cultured cells (P maker genes (PPARγ2 and MyoG) were hardly detectable at day 5, but increased significantly on day 10 (P culture system could facilitate the cells' growth and proliferation, meanwhile, inhibited the cell differentiation.

  15. Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact.

    Science.gov (United States)

    Renaud, Justine; Martinoli, Maria-Grazia

    2016-07-17

    The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

  16. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

    2008-01-01

    differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

  17. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  18. Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

    Science.gov (United States)

    Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

    2017-11-01

    Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

  19. Breast Cancer/Stromal Cells Coculture on Polyelectrolyte Films Emulates Tumor Stages and miRNA Profiles of Clinical Samples.

    Science.gov (United States)

    Daverey, Amita; Brown, Karleen M; Kidambi, Srivatsan

    2015-09-15

    In this study, we demonstrate a method for controlling breast cancer cells adhesion on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the role of tumor and stromal cell interaction on cancer biology. Numerous studies have explored engineering coculture of tumor and stromal cells predominantly using transwell coculture of stromal cells cultured onto coverslips that were subsequently added to tumor cell cultures. However, these systems imposed an artificial boundary that precluded cell-cell interactions. To our knowledge, this is the first demonstration of patterned coculture of tumor cells and stromal cells that captures the temporal changes in the miRNA signature as the breast tumor develops through various stages. In our study we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated poly(styrene) (SPS), as the polycation and polyanion, respectively, to build PEMs. Breast cancer cells attached and spread preferentially on SPS surfaces while stromal cells attached to both SPS and PDAC surfaces. SPS patterns were formed on PEM surfaces, by either capillary force lithography (CFL) of SPS onto PDAC surfaces or vice versa, to obtain patterns of breast cancer cells and patterned cocultures of breast cancer and stromal cells. In this study, we utilized cancer cells derived from two different tumor stages and two different stromal cells to effectively model a heterogeneous tumor microenvironment and emulate various tumor stages. The coculture model mimics the proliferative index (Ki67 expression) and tumor aggressiveness (HER-2 expression) akin to those observed in clinical tumor samples. We also demonstrated that our patterned coculture model captures the temporal changes in the miRNA-21 and miRNA-34 signature as the breast tumor develops through various stages. The engineered coculture platform lays groundwork toward precision medicine wherein patient-derived tumor cells can be

  20. Impact of embryo co-culture with cumulus cells on pregnancy & implantation rate in patients undergoingin vitrofertilization using donor oocyte.

    Science.gov (United States)

    Bhadarka, Harsha K; Patel, Nayana H; Patel, Niket H; Patel, Molina; Patel, Kruti B; Sodagar, Nilofar R; Phatak, Ajay G; Patel, Jagdish S

    2017-09-01

    Cumulus cell co-culture of embryo had been found to be beneficial for achieving better pregnancy and implantation rate (IR). The present study was aimed to evaluate efficiency of cumulus co-culture technique over simple culture of embryo in terms of pregnancy rate (PR) and IR in patients undergoing treatment for infertility using donor oocytes fertilized by intracytoplasmic sperm injection. This was a quasi-experimental study between control and study groups. The primary endpoint was achievement of pregnancy. Control group included 508 women who underwent embryo development without cumulus cell co-culture and study group included 394 women who underwent embryo development with cumulus cell co-culture using donor's cumulus cells. The present study demonstrated a significant increase in the IR (37.2 vs 24.2%, Pculture technique was found to be more effective than simple culture technique for embryo development in women undergoing treatment for infertility using donor oocytes fertilized by intracytoplasmic sperm injection.

  1. Comparative Neuronal Differentiation of Self-Renewing Neural Progenitor Cell Lines Obtained from Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Chiara eVerpelli

    2013-10-01

    Full Text Available Most human neuronal disorders are associated with genetic alterations that cause defects in neuronal development and induce precocious neurodegeneration. In order to fully characterize the molecular mechanisms underlying the onset of these devastating diseases, it is important to establish in vitro models able to recapitulate the human pathology as closely as possible. Here we compared three different differentiation protocols for obtaining functional neurons from human induced pluripotent stem cells (hiPSCs: human neural progenitors (hNPs obtained from hiPSCs were differentiated by co-culturing them with rat primary neurons, glial cells or simply by culturing them on matrigel in neuronal differentiation medium, and the differentiation level was compared using immunofluorescence, biochemical and electrophysiological methods.We show that the differentiated neurons displayed distinct maturation properties depending on the protocol used and the faster morphological and functional maturation was obtained when hNPs were co-cultured with rat primary neurons.

  2. Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.

    Science.gov (United States)

    Clark, Alex J; Kaller, Malte S; Galino, Jorge; Willison, Hugh J; Rinaldi, Simon; Bennett, David L H

    2017-04-01

    See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for both the development and maintenance of peripheral nerve function. We have established conditions by which human induced pluripotent stem cell-derived sensory neurons can be cultured with rat Schwann cells, and have produced for the first time long-term and stable myelinating co-cultures with human neurons. These cultures contain the specialized domains formed by axonal interaction with myelinating Schwann cells, such as clustered voltage-gated sodium channels at the node of Ranvier and Shaker-type potassium channel (Kv1.2) at the juxtaparanode. Expression of type III neuregulin-1 (TIIINRG1) in induced pluripotent stem cell-derived sensory neurons strongly enhances myelination, while conversely pharmacological blockade of the NRG1-ErbB pathway prevents myelination, providing direct evidence for the ability of this pathway to promote the myelination of human sensory axons. The β-secretase, BACE1 is a protease needed to generate active NRG1 from the full-length form. Due to the fact that it also cleaves amyloid precursor protein, BACE1 is a therapeutic target in Alzheimer's disease, however, consistent with its role in NRG1 processing we find that BACE1 inhibition significantly impairs myelination in our co-culture system. In order to exploit co-cultures to address other clinically relevant problems, they were exposed to anti-disialosyl ganglioside antibodies, including those derived from a patient with a sensory predominant, inflammatory neuropathy with mixed axonal and demyelinating electrophysiology. The co-cultures reveal that both mouse and human disialosyl antibodies target the nodal axolemma, induce acute axonal degeneration in the presence of complement, and impair myelination. The human, neuropathy-associated IgM antibody is also shown to induce complement-independent demyelination

  3. In vitro study of neural stem cells and activated Schwann cells cocultured on electrospinning polycaprolactone scaffolds

    Directory of Open Access Journals (Sweden)

    Fan BY

    2017-09-01

    Full Text Available Baoyou Fan,1,2,* Xianhu Zhou,1,2,* Lina Wang,3,* Zhijian Wei,1,2 Wei Lin,1,2 Yiming Ren,1,2 Guidong Shi,1,2 Xin Cheng,1,2 Lianyong Wang,3 Shiqing Feng,1,2 1International Science and Technology Cooperation Base of Spinal Cord Injury, Department of Orthopedic Surgery, Tianjin Medical University General Hospital, 2Tianjin Neurological Institute, Key Laboratory of Post-neuroinjury Neuro-repair and Regeneration in Central Nervous System, Ministry of Education and Tianjin City, 3Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, People’s Republic of China *These authors contributed equally to this work Background: To investigate the biocompatibility of electrospinning polycaprolactone (PCL fiber scaffolds and coculture system, which consisted of neural stem cells (NSCs and activated Schwann cells (ASCs. Materials and methods: ASCs were isolated from sciatic nerves, ligated for 7 days, in 4-week-old Wistar rats, and the NSCs were isolated from the hippocampus of E14.5 Wistar rat embryos. ASCs, NSCs and ASCs combined with NSCs were 3D cultured on the electrospinning PCL fiber scaffolds. Crystal violet staining was used to find the suitable density of ASCs for growth, and the proliferation of NSCs and ASCs were tested by Cell Counting Kit (CCK-8 assay, and cell adhesion, differentiation of NSCs and myelin basic protein (MBP expression of ASCs were observed by laser confocal microscopy. Distribution and morphology were assessed by scanning electron microscopy. Results: The average diameter of fibers in electrospinning PCL scaffolds was approximately 7.93±1.41 μm. ASCs could grow well at the density of 2×104/cm2, and a certain number of cells extended along the longitudinal axis of fibers, and the shape of the cells was spindle, which was consistent with crystal violet staining results. The CCK-8 experiment showed ASCs could proliferate gradually on the PCL scaffold within 7 days, as

  4. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19......) by a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein...... expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding...

  5. Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

    Directory of Open Access Journals (Sweden)

    Lee Myeong-Seop

    2012-04-01

    Full Text Available Abstract Backgrounds Previous studies suggested that endocrine disruptors (ED are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA and Aroclor 1254 (polychlorinated biphenyls were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. Results At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. Conclusions In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors.

  6. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Co-culture of Mouse Embryonic Stem Cells with Sertoli Cells Promote in vitro Generation of Germ Cells

    Directory of Open Access Journals (Sweden)

    Mohammad Miryounesi

    2013-06-01

    Full Text Available   Objective(s: Sertoli cells support in vivo germ cell production; but, its exact mechanism has not been well understood. The present study was designed to analyze the effect of Sertoli cells in differentiation of mouse embryonic stem cells (mESCs to germ cells.   Materials and Methods: A fusion construct composed of a Stra8 gene promoter and the coding region of enhanced green fluorescence protein was produced to select differentiated mESCs. To analyze sertoli cells’ effect in differentiation process, mESCs were separated into two groups: the first group was cultured on gelatin with retinoic acid treatment and the second group was co-cultured with sertoli cell feeder without retinoic acid induction. Expressions of pre-meiotic (Stra8, meiotic (Dazl and Sycp3 and post-meiotic (Prm1 genes were evaluated at different differentiation stages (+7, +12 and +18 days of culture. Results: In the first group, expressions of meiotic and post-meiotic genes started 12 and 18 days after induction with retinoic acid, respectively. In the second group, 7 days after co-culturing with Sertoli cells, expression of meiotic and post-meiotic genes was observed. Conclusion: These results show that differentiation process to germ cells is supported by Sertoli cells. Our findings provide a novel effective approach for generation of germ cell in vitro and studying the interaction of germ cells with their niche.

  8. The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

    Science.gov (United States)

    Duan, J.; Lu, X.; He, G.

    2017-01-01

    In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

  9. Identification of regulatory factors for mesenchymal stem cell-derived salivary epithelial cells in a co-culture system.

    Directory of Open Access Journals (Sweden)

    Yun-Jong Park

    Full Text Available Patients with Sjögren's syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05 that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.

  10. Different responses of Fe transporters in Caco2/HT29-MTX cocultures than in independent Caco-2 cell cultures

    Science.gov (United States)

    The human intestinal epithelium is composed of several cell types; mainly enterocytes and globet (mucin-secreting) cells. This study compares the cellular response for Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX coculture models for Fe bioavailability studies. Under culture, Caco-2 cell...

  11. Cell-recycle batch process of Scheffersomyces stipitis and Saccharomyces cerevisiae co-culture for second generation bioethanol production.

    Science.gov (United States)

    Ashoor, Selim; Comitini, Francesca; Ciani, Maurizio

    2015-11-01

    To achieve an optimized co-culture ratio of Scheffersomyces stipitis and Saccharomyces cerevisiae for the production of second generation bioethanol under a cell-recycle batch process. Three Sacc. cerevisiae strains were evaluated in co-culture with Sch. stipitis CBS 5773 at different ratios using synthetic medium containing glucose and xylose. Bioreactor trials indicated that the optimal condition for ethanol production using Sacc. cerevisiae EC1118 and Sch. stipitis co-culture was 1 % of O2 concentration. To increase ethanol production with Sacc. cerevisiae/Sch. stipitis co-culture a cell-recycle batch process was evaluated. Using this process, the maximum ethanol production (9.73 g l(-1)) and ethanol yield (0.42 g g(-1)) were achieved exhibiting a tenfold increase in ethanol productivity in comparison with batch process (2.1 g l(-1) h(-1)). In these conditions a stabilization of the cells ratio Sacc. cerevisiae/Sch. stipitis (1:5) at steady state condition was obtained. Batch cells recycling fermentation is an effective process to use Sch. stipitis/Sacc. cerevisiae co-culture for second generation ethanol production.

  12. Effects of barium chloride adsorbed to polyethylene glycol (PEG) microspheres on co-culture of human blood mononuclear cell and breast cancer cell lines (MCF-7).

    Science.gov (United States)

    da Silva, Fabiana Helen; Ribeiro, Aliny Aparecida Lopes; Deluque, Alessandra Lima; Cotrim, Aron Carlos de Melo; de Marchi, Patrícia Gelli Feres; França, Eduardo Luzía; Honorio-França, Adenilda Cristina

    2018-02-01

    This study investigated the effects of BaCl 2 adsorbed to polyethylene glycol (PEG) microspheres on human blood mononuclear cells (MN) co-cultured with breast cancer cell lines (MCF-7). The MCF-7 cells were obtained from the American Type Culture Collection and the blood mononuclear (MN) cells from volunteer donors. MN cells, MCF-7 cells and their co-culture (MN and MCF-7 cells) were pre-incubated for 24 h with or without 25 and 1000 pg L -1 BaCl 2 (Ba 25 and Ba 1000 ), PEG microspheres or 25 and 1000 pg L -1 BaCl 2 adsorbed to PEG microspheres (PEG-Ba 25 and PEG-Ba 1000 ). Rheological parameters and apoptosis were determined. Fluorescence microscopy and flow cytometry analyses revealed that BaCl 2 was able to adsorb the PEG microspheres. The blood flow and viscosity curves were similar among the treatments. In general, apoptosis rates increased in co-cultured cells, co-cultured cells incubated with Ba 25 and with PEG-Ba 25 , but the highest rates were observed in co-cultured cells incubated with PEG-Ba 1000 . In conclusion, BaCl 2 adsorbed to PEG microspheres exhibited dose-dependent antitumor effects against human MCF-7 breast cancer cells co-cultured with MN cells, thereby offering a possible therapeutic alternative for treating this disease provided they are administered at very low concentrations.

  13. Cell-Cell Communication Between Fibroblast and 3T3-L1 Cells Under Co-culturing in Oxidative Stress Condition Induced by H2O2.

    Science.gov (United States)

    Subramaniyan, Sivakumar Allur; Kim, Sidong; Hwang, Inho

    2016-10-01

    The present study was carried out to understand the interaction between fibroblast and 3T3-L1 preadipocyte cells under H 2 O 2 -induced oxidative stress condition. H 2 O 2 (40 μM) was added in co-culture and monoculture of fibroblast and 3T3-L1 cell. The cells in the lower well were harvested for analysis and the process was carried out for both cells. The cell growth, oxidative stress markers, and antioxidant enzymes were analyzed. Additionally, the mRNA expressions of caspase-3 and caspase-7 were selected for analysis of apoptotic pathways and TNF-α and NF-κB were analyzed for inflammatory pathways. The adipogenic marker such as adiponectin and PPAR-γ and collagen synthesis markers such as LOX and BMP-1 were analyzed in the co-culture of fibroblast and 3T3-L1 cells. Cell viability and antioxidant enzymes were significantly increased in the co-culture compared to the monoculture under stress condition. The apoptotic, inflammatory, adipogenic, and collagen-synthesized markers were significantly altered in H 2 O 2 -induced co-culture of fibroblast and 3T3-L1 cells when compared with the monoculture of H 2 O 2 -induced fibroblast and 3T3-L1 cells. In addition, the confocal microscopical investigation indicated that the co-culture of H 2 O 2 -induced 3T3-L1 and fibroblast cells increases collagen type I and type III expression. From our results, we suggested that co-culture of fat cell (3T3-L1) and fibroblast cells may influence/regulate each other and made the cells able to withstand against oxidative stress and aging. It is conceivable that the same mechanism might have been occurring from cell to cell while animals are stressed by various environmental conditions.

  14. Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Faber, Carsten; Udsen, Maja

    2012-01-01

    -cell–derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis......Purpose. To investigate the effects of T-cell–derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). Methods. We used an in vitro coculture system in which the RPE and CD3/CD28–activated T-cells were separated by a membrane. RPE cell expression...

  15. Combined strategy of endothelial cells coating, Sertoli cells coculture and infusion improves vascularization and rejection protection of islet graft.

    Directory of Open Access Journals (Sweden)

    Yang Li

    Full Text Available Improving islet graft revascularization and inhibiting rejection become crucial tasks for prolonging islet graft survival. Endothelial cells (ECs are the basis of islet vascularization and Sertoli cells (SCs have the talent to provide nutritional support and exert immunosuppressive effects. We construct a combined strategy of ECs coating in the presence of nutritious and immune factors supplied by SCs in a co-culture system to investigate the effect of vascularization and rejection inhibition for islet graft. In vivo, the combined strategy improved the survival and vascularization as well as inhibited lymphocytes and inflammatory cytokines. In vitro, we found the combinatorial strategy improved the function of islets and the effect of ECs-coating on islets. Combined strategy treated islets revealed higher levels of anti-apoptotic signal molecules (Bcl-2 and HSP-32, survival and function related molecules (PDX-1, Ki-67, ERK1/2 and Akt and demonstrated increased vascular endothelial growth factor receptor 2 (KDR and angiogenesis signal molecules (FAk and PLC-γ. SCs effectively inhibited the activation of lymphocyte stimulated by islets and ECs. Predominantly immunosuppressive cytokines could be detected in culture supernatants of the SCs coculture group. These results suggest that ECs-coating and Sertoli cells co-culture or infusion synergistically enhance islet survival and function after transplantation.

  16. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  17. Scaffold-Free Coculture Spheroids of Human Colonic Adenocarcinoma Cells and Normal Colonic Fibroblasts Promote Tumorigenicity in Nude Mice

    Directory of Open Access Journals (Sweden)

    Jong-il Park

    2016-02-01

    Full Text Available The aim of this study was to form a scaffold-free coculture spheroid model of colonic adenocarcinoma cells (CACs and normal colonic fibroblasts (NCFs and to use the spheroids to investigate the role of NCFs in the tumorigenicity of CACs in nude mice. We analysed three-dimensional (3D scaffold-free coculture spheroids of CACs and NCFs. CAC Matrigel invasion assays and tumorigenicity assays in nude mice were performed to examine the effect of NCFs on CAC invasive behaviour and tumorigenicity in 3D spheroids. We investigated the expression pattern of fibroblast activation protein-α (FAP-α by immunohistochemical staining. CAC monocultures did not form densely-packed 3D spheroids, whereas cocultured CACs and NCFs formed 3D spheroids. The 3D coculture spheroids seeded on a Matrigel extracellular matrix showed higher CAC invasiveness compared to CACs alone or CACs and NCFs in suspension. 3D spheroids injected into nude mice generated more and faster-growing tumors compared to CACs alone or mixed suspensions consisting of CACs and NCFs. FAP-α was expressed in NCFs-CACs cocultures and xenograft tumors, whereas monocultures of NCFs or CACs were negative for FAP-α expression. Our findings provide evidence that the interaction between CACs and NCFs is essential for the tumorigenicity of cancer cells as well as for tumor propagation.

  18. Role of cell division and self-propulsion in self-organization of 2D cell co-cultures

    Science.gov (United States)

    Das, Moumita; Dey, Supravat; Wu, Mingming; Ma, Minglin

    Self-organization of cells is a key process in developmental and cancer biology. The differential adhesion hypothesis (DAH), which assumes cells as equilibrium liquid droplets and relates the self-assembly of cells to differences in inter-cellular adhesiveness, has been very successful in explaining cellular organization during morphogenesis where neighboring cells have the same non-equilibrium properties (motility, proliferation rate). However, recently it has been experimentally shown that for a co-culture of two different cell types proliferating at different rates, the resulting spatial morphologies cannot be explained using the DAH alone. Motivated by this, we develop and study a two-dimensional model of a cell co-culture that includes cell division and self-propulsion in addition to cell-cell adhesion, and systemically study how cells with significantly different adhesion, motility, and proliferation rate dynamically organize themselves in a spatiotemporal and context-dependent manner. Our results may help to understand how differential equilibrium and non-equilibrium properties cooperate and compete leading to different morphologies during tumor development, with important consequences for invasion and metastasis

  19. Through gap junction communications, co-cultured mast cells and fibroblasts generate fibroblast activities allied with hypertrophic scarring.

    Science.gov (United States)

    Foley, Theodore T; Ehrlich, H Paul

    2013-05-01

    The prominent inflammatory cell identified in excessive scarring is the mast cell. Hypertrophic scar exhibits myofibroblasts derived from the transformation of fibroblasts, increased collagen synthesis, and stationary nonmigratory resident cells. The co-culture of fibroblasts with an established rat mast cell line (RMC-1) was used to explore the hypothesis of whether mast cells through gap junctional intercellular communications guide fibroblasts in promoting excessive scarring. Human dermal fibroblasts were cultured alone or co-cultured with RMC-1 cells as is or with either blocked gap junctional intercellular communications or devoid of cytoplasmic granules. Collagen synthesis was analyzed by dot blot analysis; immunohistology identified myofibroblasts, and a cell migration assay measured fibroblast locomotion. Fibroblasts co-cultured with RMC-1 cells transformed into myofibroblasts, had increased collagen synthesis, and showed retarded cell migration. In contrast, RMC-1 cells unable to form gap junctional intercellular communications were similar to fibroblasts alone, failing to promote these activities. Degranulated RMC-1 cells were as effective as intact RMC-1 cells. Mast cells induce fibroblast activities associated with hypertrophic scarring through gap junctional intercellular communications. Eliminating the mast cell or its gap junctional intercellular communications with fibroblasts may be a possible approach in preventing hypertrophic scarring or reducing fibrotic conditions.

  20. Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design.

    Science.gov (United States)

    Chen, Xiu-Min; Elisia, Ingrid; Kitts, David D

    2010-01-01

    The co-culture of Caco-2 and HT29 cells for testing intestinal drug and nutrient transport and metabolism provides the presence of both absorptive and goblet cells, both of which have different culture requirements for optimal growth and function. The research on the co-culture of Caco-2 and HT29 cells is very limited in respect to refining specific conditions that reduce intra- and inter-laboratory variations. In the present study we reported conditions that enable reproducible results to be obtained for drug permeability using in vitro co-culture of Caco-2 and HT29-MTX based on Taguchi experimental design. The selection of four factors that specified cell culture conditions, namely culture medium, seeding time, seeding density, and Caco-2:HT29-MTX ratio on TEER value and individual permeability coefficients of propranolol, ketoprofen and furosemide was established. Based on the selected conditions for co-culture, we also confirmed the functionality of the final chosen culture condition using nitric oxide as an indicator of intestinal inflammation. Choice of cell culture time and culture medium represented two of the most important factors that affected TEER values and the permeability coefficients of the model drugs. On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients. No absolute optimal cell culture condition could be obtained for all drugs; however subsequent confirmation experiments concluded that excellent precision for TEER values and drug permeability coefficients was obtained from the two operators using the following combination of conditions, namely an initial seeding density of 1 x 10(5) Caco-2 and HT29-MTX cells/cm(2) at a ratio of 9:1, followed by a 21day culture time in MEM medium. Finally, functionality of the co-culture model system using the above selected in vitro conditions resulted in comparable nitric oxide synthesis to that of a Caco-2

  1. Leukemia inhibitory factor and ciliary neurotropic factor promote the survival of Sertoli cells and gonocytes in coculture system

    NARCIS (Netherlands)

    de Miguel, M. P.; de Boer-Brouwer, M.; Paniagua, R.; van den Hurk, R.; de rooij, D. G.; van Dissel-Emiliani, F. M.

    1996-01-01

    Leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) were found to be pleiotropic modulators of Sertoli cell and gonocyte development (both isolated from the neonatal rat testis) in a coculture system, whereas IL-6, another member of this cytokine family, had no effect on these

  2. Dynamics of bone marrow-derived endothelial progenitor cell/mesenchymal stem cell interaction in co-culture and its implications in angiogenesis

    International Nuclear Information System (INIS)

    Aguirre, A.; Planell, J.A.; Engel, E.

    2010-01-01

    Research highlights: → BM-EPCs and MSCs establish complex, self-organizing structures in co-culture. → Co-culture decreases proliferation by cellular self-regulatory mechanisms. → Co-cultured cells present an activated proangiogenic phenotype. → qRT-PCR and cluster analysis identify new target genes playing important roles. -- Abstract: Tissue engineering aims to regenerate tissues and organs by using cell and biomaterial-based approaches. One of the current challenges in the field is to promote proper vascularization in the implant to prevent cell death and promote host integration. Bone marrow endothelial progenitor cells (BM-EPCs) and mesenchymal stem cells (MSCs) are bone marrow resident stem cells widely employed for proangiogenic applications. In vivo, they are likely to interact frequently both in the bone marrow and at sites of injury. In this study, the physical and biochemical interactions between BM-EPCs and MSCs in an in vitro co-culture system were investigated to further clarify their roles in vascularization. BM-EPC/MSC co-cultures established close cell-cell contacts soon after seeding and self-assembled to form elongated structures at 3 days. Besides direct contact, cells also exhibited vesicle transport phenomena. When co-cultured in Matrigel, tube formation was greatly enhanced even in serum-starved, growth factor free medium. Both MSCs and BM-EPCs contributed to these tubes. However, cell proliferation was greatly reduced in co-culture and morphological differences were observed. Gene expression and cluster analysis for wide panel of angiogenesis-related transcripts demonstrated up-regulation of angiogenic markers but down-regulation of many other cytokines. These data suggest that cross-talk occurs in between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms leading to modulation of the angiogenic response.

  3. Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

    DEFF Research Database (Denmark)

    Jantzen, Kim; Roursgaard, Martin; Madsen, Claus Desler

    2012-01-01

    Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung...... relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells....... treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine...

  4. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    International Nuclear Information System (INIS)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-01-01

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy

  5. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  6. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    Science.gov (United States)

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced

  8. Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    Directory of Open Access Journals (Sweden)

    Annalucia Carbone

    2018-01-01

    Full Text Available We previously found that human amniotic mesenchymal stem cells (hAMSCs in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ- mediated intercellular communication (GJIC in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43, a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C, and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

  9. A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution

    International Nuclear Information System (INIS)

    Yao, Rui; Zhang, Renji; Lin, Feng; Du, Yanan; Luan, Jie

    2013-01-01

    An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell–cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development. (paper)

  10. Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

    Science.gov (United States)

    2012-01-01

    Introduction The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs. Methods Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR. Results Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of

  11. Effect of Co-Culturing of Mice Liver Cells and Embryonic Carcinomatous Stem Cells on the Rate of Differentiation to Hematopoietic Cells

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    AA Pourfatollah

    2005-10-01

    Full Text Available Introduction: Considering the importance of co-culture in differentiation of embryonic stem cells, the aim of this study was evaluation of the effect of co-culturing fetal liver stroma cells with P19 cells on the line of differentiation. Materials and Methods: For this purpose, P19 cells were cultured directly in semisolid medium. These cells proliferated and primarily differentiated to colonies know as embryoid bodies (EBs after 8-12 days. The Ebs cells were trypsinized and dissociated to single or double cells. Then these cells were co-cultured on the mouse fetal liver feeder layer in the absence of exogenous factors. After 14-18 days, the colonies were studied morphologically by benzidine and giemsa staining and also counted under invert microscope. Results: The percentages of benzidine positive (or erythroid and negative colonies were 94% and 6% respectively and also the cells of colonies were studied by Giemsa staining. Results showed that they were myeloid or lymphoid type cells. Thus, the results show that in the presence of mouse fetal liver feeder layer, the number of erythroid colonies was increased. Conclusions: Therefore, this technique may be effective for differentiation of stem cells from different sources into hematopoietic cells and can be used in future for human cell therapy.

  12. Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

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    Francesca Mancuso

    2010-01-01

    Full Text Available The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM. Starting from isolated neonatal porcine pancreatic islets (NPIs, we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs for different time periods (7, 14, 21 days. To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

  13. Effects of Substrate and Co-Culture on Neural Progenitor Cell Differentiation

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    Jones, Erin Boote [Iowa State Univ., Ames, IA (United States)

    2008-01-01

    In recent years the study of stem and progenitor cells has moved to the forefront of research. Since the isolation of human hematopoietic stem cells in 1988 and the subsequent discovery of a self renewing population of multipotent cells in many tissues, many researchers have envisioned a better understanding of development and potential clinical usage in intractable diseases. Both these goals, however, depend on a solid understanding of the intracellular and extracellular forces that cause stem cells to differentiate to a specific cell fate. Many diseases of large scale cell loss have been suggested as candidates for stem cell based treatments. It is proposed that replacing the function of the damaged or defective cells by specific differentiation of stem or progenitor cells could treat the disease. Before cells can be directed to specific lineages, the mechanisms of differentiation must be better understood. Differentiation in vivo is an intensively complex system that is difficult to study. The goal of this research is to develop further understanding of the effects of soluble and extracellular matrix (ECM) cues on the differentiation of neural progenitor cells with the use of a simplified in vitro culture system. Specific research objectives are to study the differentiation of neural progenitor cells in response to astrocyte conditioned medium and protein substrate composition and concentration. In an effort to reveal the mechanism of the conditioned medium interaction, a test for the presence of a feedback loop between progenitor cells and astrocytes is presented along with an examination of conditioned medium storage temperature, which can reveal enzymatic dependencies. An examination of protein substrate composition and concentration will help to reveal the role of any ECM interactions on differentiation. This thesis is organized into a literature review covering recent advances in use of external modulators of differentiation such as surface coatings, co-culture

  14. Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal neuronal-glial networks

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    Kjell eFuxe

    2012-06-01

    Full Text Available Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT and histamine striatal afferents, the cholinergic interneurons and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal

  15. Micropatterned coculture of vascular endothelial and smooth muscle cells on layered electrospun fibrous mats toward blood vessel engineering.

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    Li, Huinan; Liu, Yaowen; Lu, Jinfu; Wei, Jiaojun; Li, Xiaohong

    2015-06-01

    A major challenge in vascular engineering is the establishment of proper microenvironment to guide the spatial organization, growth, and extracellular matrix (ECM) productions of cells found in blood vessels. In the current study, micropatterned fibrous mats with distinct ridges and grooves of different width were created to load smooth muscle cells (SMCs), which were assembled by stacking on vascular endothelial cell (EC)-loaded flat fibrous mats to mimic the in vivo-like organized structure of blood vessels. SMCs were mainly distributed in the ridges, and aligned fibers in the patterned regions led to the formation of elongated cell bodies, intense actin filaments, and expressions of collagen I and α-smooth muscle actin in a parallel direction with fibers. ECs spread over the flat fibrous mats and expressed collagen IV and laminin with a cobblestone-like feature. A z-stack scanning of fluorescently stained fibrous mats indicated that SMCs effectively infiltrated into fibrous scaffolds at the depth of around 200 μm. Compared with SMCs cultured alone, the coculture with ECs enhanced the proliferation, infiltration, and cytoskeleton elongation of SMCs on patterned fibrous mats. Although the coculture of SMCs made no significant difference in the EC growth, the coculture system on patterned fibrous scaffolds promoted ECM productions of both ECs and SMCs. Thus, this patterned fibrous configuration not only offers a promising technology in the design of tissue engineering scaffolds to construct blood vessels with durable mechanical properties, but also provides a platform for patterned coculture to investigate cell-matrix and cell-cell interactions in highly organized tissues. © 2014 Wiley Periodicals, Inc.

  16. Damage of neuroblastoma cell SH-SY5Y mediated by MPP+ inhibits proliferation of T-cell leukemia Jurkat by co-culture system.

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    Wang, Fuli; Awan, Umer Farooq; Wang, Yuanyuan; Wang, Luna; Qing, Hong; Ma, Hong; Deng, Yulin

    2014-06-13

    The adaptive immune system has implications in pathology of Parkinson's disease (PD). Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+) for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM) arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2) and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM) induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

  17. Damage of Neuroblastoma Cell SH-SY5Y Mediated by MPP+ Inhibits Proliferation of T-Cell Leukemia Jurkat by Co-Culture System

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    Fuli Wang

    2014-06-01

    Full Text Available The adaptive immune system has implications in pathology of Parkinson’s disease (PD. Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+ for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2 and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells.

  18. Non-Viable Lactobacillus Casei Beneficially Modulates Poly I:C Immune Response in Co-Cultures of Human Cells.

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    Vintiñi, Elisa; Medina, Marcela

    2017-12-01

    Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viral stimulus to mimic in vivo and in vitro infection induced by some viruses. To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture models of peripheral blood mononuclear cells (PBMC) and A549 cells. T and NK cell activation was evaluated by flow cytometry and levels of TNF-α, IFN-γ, IL-10, IL-29, and IL-17 by ELISA. Cells in direct contact with A549 (PBMC-A549) and cells with no contact with it (PBMC//A549) were used for this purpose. PBMCs alone and both co-culture systems were stimulated for 24 h with the following stimuli: LPS (10 µg/ml), LcM (106 UFC/ml), Poly I:C (2 µg/ml), Poly I:C+LcM, and LcM (3 h)+Poly I:C. Moreover, unstimulated cells were used as a control. Poly I:C and LcM (3 h)+Poly I:C in PBMC-A549 showed a significant increase in the percentage of CD8+ expression (psystem. However, activation percentages were higher in direct co-culture. Poly I:C induced a higher level of pro-inflammatory TNF-α and IFN-γ cytokines as well as IL-17 and IL-29 with lower IL-10 levels in both co-culture systems while LcM induced a beneficial pattern of cytokines that would regulate Poly I:C effect. This in vitro model allowed us to highlight the potential of LcM as a modulator of anti-viral immune response and suggest its potential use in formulations against RNA respiratory viruses.

  19. Pellet coculture of osteoarthritic chondrocytes and infrapatellar fat pad-derived mesenchymal stem cells with chitosan/hyaluronic acid nanoparticles promotes chondrogenic differentiation

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    Shu Huang

    2017-11-01

    Full Text Available Abstract Background Cell source plays a key role in cell-based cartilage repair and regeneration. Recent efforts in cell coculture have attempted to combine the advantages and negate the drawbacks of the constituent cell types. The aim of this study was to evaluate the chondrogenic outcome of articular chondrocytes (ACs and infrapatellar fat pad (IPFP-derived mesenchymal stem cells (MSCs in direct coculture. Methods ACs and IPFP MSCs from the same patients with knee osteoarthritis (OA were cocultured in monolayer and in pellets. The monocultures of each cell type were also used as controls. Morphological and histologic analysis, immunofluorescence staining, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay were performed to characterize the chondrogenic differentiation of cocultures. Furthermore, the effects of chitosan/hyaluronic acid (CS/HA nanoparticle exposure on the chondrogenesis of cocultures were examined. Results In both monolayer and pellet coculture, the hypertrophy of MSCs and the inflammatory activities of ACs were inhibited, although the chondrogenic production in coculture was not promoted compared with that in monoculture. In addition, the exposure of CS/HA nanoparticles to pellet coculture improved the production of type II collagen and aggrecan. Conclusions We demonstrate for the first time that pellet coculture of ACs and IPFP MSCs with CS/HA nanoparticles could promote chondrogenic outcome while preventing the inflammatory status of ACs and the hypertrophic differentiation of MSCs. These findings suggest that the combination of ACs, IPFP MSCs, and CS/HA might be useful in cartilage repair in knee OA.

  20. Coculture with endothelial cells enhances osteogenic differentiation of periodontal ligament stem cells via cyclooxygenase-2/prostaglandin E2/vascular endothelial growth factor signaling under hypoxia.

    Science.gov (United States)

    Zhao, Lixing; Wu, Yeke; Tan, Lijun; Xu, Zhenrui; Wang, Jun; Zhao, Zhihe; Li, Xiaoyu; Li, Yu; Yang, Pu; Tang, Tian

    2013-12-01

    During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.

  1. Chondrogenesis of human adipose derived stem cells for future microtia repair using co-culture technique.

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    Goh, Bee See; Che Omar, Siti Nurhadis; Ubaidah, Muhammad Azhan; Saim, Lokman; Sulaiman, Shamsul; Chua, Kien Hui

    2017-04-01

    In conclusion, these result showed HADSCs could differentiate into chondrocytes-like cells, dependent on signaling induced by TGF-β3 and chondrocytes. This is a promising result and showed that HADSCs is a potential source for future microtia repair. The technique of co-culture is a positive way forward to assist the microtia tissue. Reconstructive surgery for the repair of microtia still remains the greatest challenge among the surgeons. Its repair is associated with donor-site morbidity and the degree of infection is inevitable when using alloplastic prosthesis with uncertain long-term durability. Thus, human adipose derived stem cells (HADSCs) can be an alternative cell source for cartilage regeneration. This study aims to evaluate the chondrogenic potential of HADSCs cultured with transforming growth factor-beta (TGF-β) and interaction of auricular chondrocytes with HADSCs for new cartilage generation. Multi-lineages differentiation features of HADSCs were monitored by Alcian Blue, Alizarin Red, and Oil Red O staining for chondrogenic, adipogenic, and osteogenic differentiation capacity, respectively. Further, HADSCs alone were culture in medium added with TGF-β3; and human auricular chondrocytes were interacted indirectly in the culture with and without TGF-βs for up to 21 days, respectively. Cell morphology and chondrogenesis were monitored by inverted microscope. For cell viability, Alamar Blue assay was used to measure the cell viability and the changes in gene expression of auricular chondrocyte markers were determined by real-time polymerase chain reaction analysis. For the induction of chondrogenic differentiation, HADSCs showed a feature of aggregation and formed a dense matrix of proteoglycans. Staining results from Alizirin Red and Oil Red O indicated the HADSCs also successfully differentiated into adipogenic and osteogenic lineages after 21 days. According to a previous study, HADSCs were strongly positive for the mesenchymal markers CD90, CD73

  2. Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

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    Ting Shen

    2017-05-01

    Full Text Available AIM: To investigate the impact of adipose-derived mesenchymal stem cells (ADSCs on cell viability and extracellular matrix (ECM synthesis of corneal stromal cells (CSCs. METHODS: ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly co-cultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate (FITC/proliferation indices (PI assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase (MMP, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS: ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis (early and late between the negative control group (6.34% and 2.06% and the ADCSs-treated group (4.69% and 1.59%. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type (I, II, III, IV, and V in CSCs were observed in CSCs/ADSCs group, 3.47-fold, 4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models. CONCLUSION: ADSCs play a promotive role in CSCs’ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation

  3. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

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    Hidenori Otsuka, Kohei Sasaki, Saya Okimura, Masako Nagamura and Yuichi Nakasone

    2013-01-01

    Full Text Available Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol (PEG hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (phgr = 100 μm were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts or endothelial cells (BAECs: bovine aortic endothelial cells were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

  4. Temporally coordinated spiking activity of human induced pluripotent stem cell-derived neurons co-cultured with astrocytes.

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    Kayama, Tasuku; Suzuki, Ikuro; Odawara, Aoi; Sasaki, Takuya; Ikegaya, Yuji

    2018-01-01

    In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Co-culture with NK-92MI cells enhanced the anti-cancer effect of bee venom on NSCLC cells by inactivation of NF-κB.

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    Kollipara, Pushpa Saranya; Kim, Jung Hyun; Won, Dohee; Lee, Sang Min; Sung, Ha Chang; Chang, Hyun Sok; Lee, Kang Tae; Lee, Kang Sik; Park, Mi Hee; Song, Min Jong; Song, Ho Sueb; Hong, Jin Tae

    2014-03-01

    In the present study we experimented on a multimodal therapeutic approach, such as combining chemotherapy agent (Bee venom) with cellular (NK-92MI) immunotherapy. Previously bee venom has been found to show anti-cancer effect in various cancer cell lines. In lung cancer cells bee venom showed an IC(50) value of 3 μg/ml in both cell lines. The co-culture of NK-92MI cell lines with lung cancer cells also show a decrease in viability upto 50 % at 48 h time point. Hence we used bee venom treated NK-92MI cells to co-culture with NSCLC cells and found that there is a further decrease in cell viability upto 70 and 75 % in A549 and NCI-H460 cell lines respectively. We further investigated the expression of various apoptotic and anti-apoptotic proteins and found that Bax, cleaved caspase-3 and -8 were increasing where as Bcl-2 and cIAP-2 was decreasing. The expression of various death receptor proteins like DR3, DR6 and Fas was also increasing. Concomitantly the expression of various death receptor ligands (TNFalpha, Apo3L and FasL) was also increasing of NK-92MI cells after co-culture. Further the DNA binding activity and luciferase activity of NF-κB was also inhibited after co-culture with bee venom treated NK-92MI cell lines. The knock down of death receptors with si-RNA has reversed the decrease in cell viability and NF-κB activity after co-culture with bee venom treated NK-92MI cells. Thus this new approach can enhance the anti-cancer effect of bee venom at a much lower concentration.

  6. Impact of commercial cigarette smoke condensate on brain tissue co-cultured with astrocytes and blood-brain barrier endothelial cells.

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    Lee, Seon-Bong; Kim, Ju-Hyeong; Cho, Myung-Haing; Choe, Eun-Sang; Kim, Kwang-Sik; Shim, Soon-Mi

    2017-01-01

    The purpose of the current study was to investigate the effect of two commercial cigarette smoke condensates (CCSC) on oxidative stress and cell cytotoxicity in human brain (T98G) or astrocytes (U-373 MG) in the presence of human brain microvascular endothelial cells (HBMEC). Cell viability of mono-culture of T98G or U-373 MG was markedly decreased in a concentration-dependent manner, and T98G was more susceptible than U-373 MG to CCSC exposure. Cytotoxicity was less prominent when T98G was co-cultured with HBMEC than when T98G was co-cultured with U-373 MG. Significant reduction in trans-epithelial electric resistance (TEER), a biomarker of cellular integrity was noted in HBMEC co-cultured with T98G (HBMEC-T98G co-culture) and U-373 MG co-cultured with T98G (U-373 MG-T98G co-culture) after 24 or 48 hr CCSC exposure, respectively. TEER value of U-373 MG co-cultured with T98G (79-84%) was higher than HBMEC co-cultured with T98G (62-63%) within 120-hr incubation with CCSC. Reactive oxygen species (ROS) generated by CCSC in mono-culture of T98G and U-373 MG reached highest levels at 4 and 16 mg/ml, respectively. ROS production by T98G fell when co-cultured with HBMEC or U-373MG. These findings suggest that adverse consequences of CCSC treatment on brain cells may be protected by blood-brain barrier or astrocytes, but with chronic exposure toxicity may be worsened due to destruction of cellular integrity.

  7. 3D printed scaffolds of calcium silicate-doped β-TCP synergize with co-cultured endothelial and stromal cells to promote vascularization and bone formation.

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    Deng, Yuan; Jiang, Chuan; Li, Cuidi; Li, Tao; Peng, Mingzheng; Wang, Jinwu; Dai, Kerong

    2017-07-17

    Synthetic bone scaffolds have potential application in repairing large bone defects, however, inefficient vascularization after implantation remains the major issue of graft failure. Herein, porous β-tricalcium phosphate (β-TCP) scaffolds with calcium silicate (CS) were 3D printed, and pre-seeded with co-cultured human umbilical cord vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs) to construct tissue engineering scaffolds with accelerated vascularization and better bone formation. Results showed that in vitro β-TCP scaffolds doped with 5% CS (5%CS/β-TCP) were biocompatible, and stimulated angiogenesis and osteogenesis. The results also showed that 5%CS/β-TCP scaffolds not only stimulated co-cultured cells angiogenesis on Matrigel, but also stimulated co-cultured cells to form microcapillary-like structures on scaffolds, and promoted migration of BMSCs by stimulating co-cultured cells to secrete PDGF-BB and CXCL12 into the surrounding environment. Moreover, 5%CS/β-TCP scaffolds enhanced vascularization and osteoinduction in comparison with β-TCP, and synergized with co-cultured cells to further increase early vessel formation, which was accompanied by earlier and better ectopic bone formation when implanted subcutaneously in nude mice. Thus, our findings suggest that porous 5%CS/β-TCP scaffolds seeded with co-cultured cells provide new strategy for accelerating tissue engineering scaffolds vascularization and osteogenesis, and show potential as treatment for large bone defects.

  8. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

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    Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young; Yeo, Jee Eun; Choi, Yun-Jin; Kim, Yong Il; Koh, Yong-Gon, E-mail: yonseranglab@daum.net

    2014-05-16

    Highlights: • Co-culture of hSDMSCs with SNP-stimulated chondrocytes improves anti-inflammation. • Co-culture system produces IGF-1. • Co-culture system suppresses inflammatory genes expression. • Co-culture system improves cell proliferation. • Exogenous IGF-1 inhibits inflammatory activity in SNP-stimulated chondrocytes. - Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culture of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.

  9. Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts

    International Nuclear Information System (INIS)

    Samoszuk, Michael; Tan, Jenny; Chorn, Guillaume

    2005-01-01

    Accumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts. We measured the clonogenic growth of small numbers of human breast cancer cells co-cultured in direct contact with serum-activated, normal human fibroblasts. Using DNA microarrays, we also characterized the gene expression profile of the serum-activated fibroblasts. In order to validate the in vivo relevance of our experiments, we then analyzed clinical samples of metastatic breast cancer for the presence of myofibroblasts expressing α-smooth muscle actin. Clonogenic growth of human breast cancer cells obtained directly from in situ and invasive tumors was dramatically and consistently enhanced when the tumor cells were co-cultured in direct contact with serum-activated fibroblasts. This effect was abolished when the cells were co-cultured in transwells separated by permeable inserts. The fibroblasts in our experimental model exhibited a gene expression signature characteristic of 'serum response' (i.e. myofibroblasts). Immunostaining of human samples of metastatic breast cancer tissue confirmed that myofibroblasts are in direct contact with breast cancer cells. Serum-activated fibroblasts promote the clonogenic growth of human breast cancer cells in vitro through a mechanism that involves direct physical contact between the cells. This model shares many important molecular and phenotypic similarities with the fibroblasts that are naturally found in breast cancers

  10. Nicotine and caffeine alter the effects of the LPS- primed mesenchymal stem cells on the co-cultured neutrophils.

    Science.gov (United States)

    Abbasi, Ardeshir; Kukia, Nasim Rahmani; Froushani, Seyyed Meysam Abtahi; Hashemi, Seyed Mahmoud

    2018-04-15

    Mesenchymal stem cells (MSCs) express some of the nicotinic receptor subunits and adenosine receptors. The communication between tissue MSCs with neutrophils has been shown in previous studies. The aim of the present study is to determine the role of nicotine or caffeine on MSCs and its effects on neutrophils. After the isolation, MSCs were pulsed with LPS (10 ng/ml) for 1 h. Then, MSCs were incubated with different concentrations of caffeine (0.1, 0.5 and 1 mM) and or with different concentrations of nicotine (0.1, 0.5, and 1 μM) for 48 h. Afterwards, the medium was aspirated and the cells were used for co-culture experiment with neutrophil. The obtained data showed that LPS primed MSCs could decrease neutrophil vitality, whereas the treatment of MSCs with nicotine and/or especially a treatment with caffeine reverse this effect. Obtained data showed that when the LPS-primed MSCs were treated with nicotine or caffeine, the vitality of co-cultured neutrophils was significantly increased. The rate of the respiratory burst of neutrophils after co-culture by LPS-primed MSCs was decreased compared to the respiratory burst of neutrophil alone. Nicotine and/or caffeine treatment could reverse this reduction. Generally, these findings provide a new insight into understanding the anti-inflammatory and immunomodulatory effects of nicotine and caffeine. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering

    DEFF Research Database (Denmark)

    Wenger, Andreas; Kowalewski, Nadja; Stahl, Andreas

    2005-01-01

    and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit...... to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due...

  12. Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system.

    Science.gov (United States)

    Fisher, Bridget S; Estraño, Carlos E; Cole, Judith A

    2013-01-01

    Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.

  13. Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique.

    Science.gov (United States)

    Noruddin, Nur Adelina Ahmad; Saim, Aminuddin B; Chua, Kien Hui; Idrus, Ruszymah

    2007-12-01

    To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application. Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells. Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques. Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.

  14. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

    Science.gov (United States)

    Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

    2012-01-01

    In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

  15. The chalcone butein from Rhus verniciflua Stokes inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts

    Directory of Open Access Journals (Sweden)

    Tan Jenny

    2005-03-01

    Full Text Available Abstract Background Butein (3,4,2',4'-tetrahydroxychalone, a plant polyphenol, is a major biologically active component of the stems of Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro. Methods We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL or various other modulators of fibroblast function (troglitazone-1 μg/mL; GW9662-1 μM; meloxican-1 μM; and 3,4 dehydroproline-10 μg/mL. In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL. Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL. Results Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also

  16. In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients.

    Science.gov (United States)

    Riboldi, Marcia; Rubio, Carmen; Pellicer, Antonio; Gil-Salom, Manuel; Simón, Carlos

    2012-09-01

    To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells. Prospective analysis. Research center. Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients. TESE, with enzymatic dissociation of samples to obtain a cell suspension, which was cultured for 4 days with 4 ng/mL GDNF. The CD49f+ cells were sorted using fluorescence-activated cell sorting (FACS) as a marker to identify spermatogonial stem cells (SSCs), which were cocultured with Sertoli cells expressing red fluorescent protein (RFP) in knockout serum replacement (KSR) media with addition of 1,000 IU/mL of follicle-stimulating hormone (FSH), 1 μM testosterone, 40 ng/mL of GDNF, and 2 μM retinoic acid (RA) for 15 days in culture at 37°C and 5% CO(2) to induce meiotic progression. Cells were collected and analyzed by immunofluorescence for meiosis progression with specific markers SCP3 and CREST, and they were confirmed by fluorescence in situ hybridization (FISH). Isolation of CD49f+ cells and coculture with Sertoli cells, meiosis progression in vitro, assessment of SSCs and meiotic markers real-time polymerase chain reaction (RT-PCR), immunohistochemical analysis, and FISH. The CD49f+ isolated from the of total cell count in the TESE samples of azoospermic patients varied from 5.45% in OA to 2.36% in NOA. Sertoli cells were obtained from the same TESE samples, and established protocols were used to characterize them as positive for SCF, rGDNF, WT1, GATA-4, and vimentin, with the presence of tight junctions and lipid droplets shown by oil red staining. After isolation, the CD49f+ cells were cocultured with RFP Sertoli cells in a 15-day time-course experiment. Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from one NOA patient. A FISH analysis for chromosomes 13, 18, 21, X, and Y confirmed the presence of haploid cells on day

  17. A simple coculture system shows mutualism between anaerobic faecalibacteria and epithelial Caco-2 cells

    NARCIS (Netherlands)

    Sadaghian, Mehdi; von Martels, Julius Z. H.; Khan, Muhammad; Blokzijl, Tjasso; Paglia, Giuseppe; Dijkstra, Gerard; Harmsen, Hermie J. M.; Faber, Klaas Nico

    2015-01-01

    Most gut bacteria are obligate anaerobes and are important for human health. However, little mechanistic insight is available on the health benefits of specific anaerobic gut bacteria. A main obstacle in generating such knowledge is the lack of simple and robust coculturing methods for anaerobic

  18. Human Wharton’s jelly-derived mesenchymal stem cells express oocyte developmental genes during co-culture with placental cells

    Directory of Open Access Journals (Sweden)

    Hamid Reza Asgari

    2015-01-01

    Conclusion: Placental cell supplementsTransforming growth factor (TGF α, β and basic fibroblast growth factor (bFGF in a co-culture model can provide proper environment for induction of HUMSCs into PGCs and expression of oocyte-like markers.

  19. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device

    NARCIS (Netherlands)

    van der Meer, Andries Dirk; Orlova, Valeria V.; tem Dijke, Peter; van den Berg, Albert; Mummery, Christine L.

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels

  20. Human Endothelial-Like Differentiated Precursor Cells Maintain Their Endothelial Characteristics When Cocultured with Mesenchymal Stem Cell and Seeded onto Human Cancellous Bone

    Directory of Open Access Journals (Sweden)

    Dirk Henrich

    2013-01-01

    Full Text Available Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC’s collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter’s collagen-1α gene expression.

  1. Uniaxial cyclic strain of human adipose–derived mesenchymal stem cells and C2C12 myoblasts in coculture

    Directory of Open Access Journals (Sweden)

    James M Dugan

    2014-03-01

    Full Text Available Tissue engineering skeletal muscle in vitro is of great importance for the production of tissue-like constructs for treating tissue loss due to traumatic injury or surgery. However, it is essential to find new sources of cells for muscle engineering as efficient in vitro expansion and culture of primary myoblasts are problematic. Mesenchymal stem cells may be a promising source of myogenic progenitor cells and may be harvested in large numbers from adipose tissue. As skeletal muscle is a mechanically dynamic tissue, we have investigated the effect of cyclic mechanical strain on the myogenic differentiation of a coculture system of murine C2C12 myoblasts and human adipose–derived mesenchymal stem cells. Fusion of mesenchymal stem cells with nascent myotubes and expression of human sarcomeric proteins was observed, indicating the potential for myogenic differentiation of human mesenchymal stem cells. Cyclic mechanical strain did not affect the fusion of mesenchymal stem cells, but maturation of myotubes was perturbed.

  2. Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering

    Directory of Open Access Journals (Sweden)

    He X

    2015-03-01

    Full Text Available Xiaomin He,1,* Bei Feng,1,2,* Chuanpei Huang,1 Hao Wang,1 Yang Ge,1 Renjie Hu,1 Meng Yin,1 Zhiwei Xu,1 Wei Wang,1 Wei Fu,1,2 Jinghao Zheng1 1Department of Pediatric Cardiothoracic Surgery, 2Institute of Pediatric Translational Medicine, Shanghai Children’s Medical Center School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC/chondrocyte cocultures (75% BMSCs and 25% chondrocytes in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young’s modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs. Keywords: electrospinning, nanocomposite, cartilage tissue engineering, nanomaterials, stem cells

  3. Assessment of cellular materials generated by co-cultured ‘inflamed’ and healthy periodontal ligament stem cells from patient-matched groups

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Hao-Ning [State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The First Affiliated Hospital of the Chinese PLA General Hospital, Beijing 100048 (China); Xia, Yu [State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 309th Hospital of Chinese People' s Liberation Army, Beijing 100091 (China); Xu, Jie; Tian, Bei-Min; Zhang, Xi-Yu [State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical University, Xi' an (China); Chen, Fa-Ming, E-mail: cfmsunhh@fmmu.edu.cn [State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Periodontology, School of Stomatology, Fourth Military Medical University, Xi' an (China)

    2016-08-01

    Recently, stem cells derived from the'inflamed’ periodontal ligament (PDL) tissue of periodontally diseased teeth (I-PDLSCs) have been increasingly suggested as a more readily accessible source of cells for regenerative therapies than those derived from healthy PDL tissue (H-PDLSCs). However, substantial evidence indicates that I-PDLSCs exhibit impaired functionalities compared with H-PDLSCs. In this study, patient-matched I-PDLSCs and H-PDLSCs were co-cultured at various ratios. Cellular materials derived from these cultures were investigated regarding their osteogenic potential in vitro and capacity to form new bone following in vivo transplantation. While patient-matched I-PDLSCs and H-PDLSCs could co-exist in co-culture systems, the proportion of I-PDLSCs tended to increase during in vitro incubation. Compared with H-PDLSC monoculture, the presence of I-PDLSCs in the co-cultures appeared to enhance the overall cell proliferation. Although not completely rescued, the osteogenic and regenerative potentials of the cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Notably, cells in co-cultures containing either 50% I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs expressed osteogenesis-related proteins and genes at levels similar to those expressed in H-PDLSC monocultures (P>0.05). Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures. These data suggest that the co-culture of I-PDLSCs with patient-matched H-PDLSCs is a practical and effective method for increasing the overall osteogenic and regenerative potentials of resultant cellular materials. - Highlights: • Co-culturing H-PDLSCs with I-PDLSCs led to rapid cell expansion. • H-PDLSCs and I-PDLSCs co-cultured

  4. Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

    Science.gov (United States)

    Futrega, Kathryn; Atkinson, Kerry; Lott, William B; Doran, Michael R

    2017-04-01

    While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34 + progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34 + cell expansion outcomes. By contrast, a substantial increase in CD34 + CD38 - cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34 + CD38 - cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34 + CD38 - cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34 + cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

  5. Human umbilical cord-derived mesenchymal stem cells differentiate into epidermal-like cells using a novel co-culture technique.

    Science.gov (United States)

    Li, Dongjie; Chai, Jiake; Shen, Chuanan; Han, Yanfu; Sun, Tianjun

    2014-08-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) isolated from human umbilical Wharton's Jelly are a population of primitive and pluripotent cells. In specific conditions, hUCMSCs can differentiate into various cells, including adipocytes, osteoblasts, chondrocytes, neurocytes, and endothelial cells. However, few studies have assessed their differentiation into epidermal cells in vitro. To assess the potential of hUCMSCs to differentiate into epidermal cells, a microporous membrane-based indirect co-culture system was developed in this study. Epidermal stem cells (ESCs) were seeded on the bottom of the microporous membrane, and hUCMSCs were seeded on the top of the microporous membrane. Cell morphology was assessed by phase contrast microscopy, and the expression of early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), and β1-integrin, was determined by immunofluorescence, Western blot, and quantitative real-time PCR (Q-PCR) analyses. hUCMSC morphology changed from spindle-like to oblate or irregular with indirect co-culture with ESCs; they also expressed greater levels P63, CK19, and β1-integrin mRNA and protein compared to the controls (p cultures, indirect co-culture expressed significantly greater CK19 protein (p culture model.

  6. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  7. A microfluidic hepatic coculture platform for cell-based drug metabolism studies.

    Science.gov (United States)

    Novik, Eric; Maguire, Timothy J; Chao, Piyun; Cheng, K C; Yarmush, Martin L

    2010-04-01

    Within the global pharmaceutical and biotech industries, there is significant interest in identifying in vitro screening systems that are more human-relevant-i.e., that offer greater utility in predicting subcellular and cellular physiological responses in humans in vivo-and that thereby allow investigators to reduce the incidence of costly late-stage failures during pharmaceutical clinical trials, as well as to reduce the use of animals in drug testing. Currently incumbent in vitro screening methods, such as culturing human hepatocytes in suspension, while useful, are limited by a lack of long term cellular function. In order to address this limitation, we have established an integrated, microfluidic, in vitro platform that combines the patented HmuREL((R)) microdevice with a hepatic coculture system. In the present report, we use this platform to study clearance and metabolite generation of a battery of molecular entities. The results show that the flow-based coculture system is capable of clearing, with improved resolution and predictive value, compounds with high, medium, and low clearance values. In addition, when coculture is coupled with flow, higher metabolite production rates are obtained than in static systems. Copyright 2009 Elsevier Inc. All rights reserved.

  8. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Haller, D.; Brinz, S.

    2004-01-01

    To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model...... was investigated. Nonpathogenic Escherichia coli were added either to the apical or the basolateral compartment of this transwell cell culture system, the latter of which contained human leucocytes. The synthesis of tumour necrosis factor (TNF-alpha) and interleukin (IL)-12 was significantly suppressed by CACO-2...... cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8...

  9. Direct binding of boar ejaculate and epididymal spermatozoa to porcine epididymal epithelial cells is also needed to maintain sperm survival in in vitro co-culture.

    Science.gov (United States)

    Yeste, Marc; Castillo-Martín, Míriam; Bonet, Sergi; Briz, Maria Dolors

    2012-04-01

    The aim of the present study was to compare the influence of cultured epididymal epithelial cells (EEC) from corpus, caput or cauda, oviductal epithelial cells (OEC) and non-reproductive epithelial cells (LLC-PK1) on function and survival of epididymal and ejaculated spermatozoa, in the latter case to determine whether such influence differed between morphologically normal and abnormal spermatozoa. For this purpose, either spermatozoa were directly co-cultured with EEC from caput, corpus, or cauda, OEC and LLC-PK1 cells (experiment 1) or a membrane-diffusible insert was included in these co-cultures (experiment 2). EEC cultured from the three epididymal regions did not differently affect the sperm parameters. Morphologically normal spermatozoa presented a higher ability to bind EEC, OEC, and LLC-PK1 than abnormal spermatozoa with cytoplasmic droplets or with tail/head malformations. Epididymal spermatozoa were more able to bind EEC during the first 24 h of co-culture, while ejaculated spermatozoa presented a higher capacity to bind OEC between 30 min and 3 h of co-incubation. In all cases, the ability to bind to epithelial cells was higher when they were co-cultured with EEC and OEC than with LLC-PK1. After 2 h of co-culture, the viability of epididymal spermatozoa was better maintained when they bound EEC than when they bound OEC. Conversely, the viability of ejaculated spermatozoa was better maintained when bound OEC than when bound EEC after 24 and 48 h of co-culture. Our work, apart from corroborating the involvement of morphologically normal spermatozoa in the formation of sperm reservoir, highlights the importance of direct contact spermatozoa-EEC in maintaining the sperm survival in in vitro co-culture, and also suggests that a specific binding between EEC and epididymal spermatozoa exists. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Analysis of the CD8+T cell anti-HIV activity in heterologous cell co-cultures reveals the benefit of multiple HLA class I matches.

    Science.gov (United States)

    Killian, M Scott; Teque, Fernando; Sudhagoni, Ramu

    2018-02-01

    CD8 + T lymphocytes can reduce the production of human immunodeficiency virus 1 (HIV-1) by CD4 + T cells by cytotoxic and non-cytotoxic mechanisms. To investigate the involvement of human leukocyte antigen (HLA) class I compatibility in anti-HIV responses, we co-cultured primary CD8 + T cells, isolated from the peripheral blood of HIV-1-infected individuals, with panels of autologous and heterologous acutely HIV-1-infected primary CD4 + T cells. Altogether, CD8 + T cell anti-HIV activity was evaluated in more than 200 co-cultures. Marked heterogeneity in HIV-1 replication levels was observed among the co-cultures sharing a common CD8 + T cell source. The co-cultures that exhibited greater than 50% reduction in HIV production were found to have significantly increased numbers of matching HLA class I alleles (Yates chi-square = 54.21; p CD8 + T cells from HIV controllers and asymptomatic viremic individuals, matching HLA-B and/or HLA-C alleles were more predictive of strong anti-HIV activity than matching HLA-A alleles. Overall, HLA class I genotype matches were more closely associated with CD8 + T cell anti-HIV activity than supertype pairings. Antibodies against HLA class I and CD3 reduced the CD8 + T cell anti-HIV activity. Stimulated CD8 + T cells exhibited increased anti-HIV activity and reduced dependency on HLA compatibility. These findings provide evidence that the maximal suppression of HIV replication by CD8 + T cells requires the recognition of multiple epitopes. These studies provide insight for HIV vaccine development, and the analytic approach can be useful for the functional characterization of HLA class I alleles and tentative HLA class I supertypes.

  11. Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

    Science.gov (United States)

    Rath, Subha N.; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E.; Boccaccini, Aldo R.; Kneser, Ulrich

    2014-01-01

    Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID

  12. Bioactive copper-doped glass scaffolds can stimulate endothelial cells in co-culture in combination with mesenchymal stem cells.

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    Subha N Rath

    Full Text Available Bioactive glass (BG scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC. In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture. Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP assay or by PCR, there was increased vascular endothelial growth factor (VEGF expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering

  13. Differentiation of Mouse Ovarian Stem Cells Toward Oocyte-Like Structure by Coculture with Granulosa Cells.

    Science.gov (United States)

    Parvari, Soraya; Yazdekhasti, Hossein; Rajabi, Zahra; Gerayeli Malek, Valliollah; Rastegar, Tayebeh; Abbasi, Mehdi

    2016-11-01

    An increasing body of evidence has confirmed existence and function of ovarian stem cells (OSCs). In this study, a novel approach on differentiation of OSCs into oocyte-like cells (OLCs) has been addressed. Recently, different methods have been recruited to isolate and describe aspects of OSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate OSCs. Cell suspension of mouse neonatal ovaries was cultured and formed colonies were harvested mechanically and cultivated on mouse embryonic fibroblasts. For differentiation induction, colonies transferred on inactive granulosa cells. Results showed that cells in colonies were positive for alkaline phosphatase activity and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the pluripotency characteristics of cells. Immunofluorescence revealed a positive signal for OCT4, DAZL, MVH, and SSEA1 in colonies as well. Results of RT-PCR and immunofluorescence confirmed that some OLCs were generated within the germ stem cell (GSCs) colonies. The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economic than other techniques. Our results demonstrate that granulosa cells were effective in inducing the differentiation of OSCs into OLCs through direct cell-to-cell contacts.

  14. Amniotic mesenchymal stem cells mitigate osteoarthritis progression in a synovial macrophage-mediated in vitro explant coculture model.

    Science.gov (United States)

    Topoluk, Natasha; Steckbeck, Kathleen; Siatkowski, Sandra; Burnikel, Brian; Tokish, John; Mercuri, Jeremy

    2017-11-13

    Osteoarthritis (OA) is a disease of the synovial joint marked by chronic, low-grade inflammation leading to cartilage destruction. Regenerative medicine strategies for mitigating OA progression and/or promoting cartilage regeneration must be assessed using models that mimic the hallmarks of OA. More specifically, these models should maintain synovial macrophage phenotype in their native micro-environment. Herein, an in vitro coculture model of patient-matched human OA cartilage and synovium was assessed for viability, macrophage phenotype, and progressive cartilage destruction in the presence of an inflammatory milieu. Additionally, the influence of synovial macrophages and their polarization within the model was defined using depletion studies. Finally, the model was used to compare the ability of human amniotic stem cells (hAMSCs) and human adipose stem cells (hADSCs) to mitigate OA progression. OA cocultures demonstrated progressive and significant reductions in chondrocyte viability and cartilage glycosaminoglycan content within a proinflammatory environment. Selective depletion of synovial macrophages resulted in significant decreases in M1:M2 percentage ratio yielding significant reductions in concentrations of interleukin-1 beta, matrix metalloproteinase-13 and attenuation of cartilage damage. Finally, hAMSCs were found to be more chondroprotective versus hADSCs as indicated by significantly improved OA chondrocyte viability (89.8 ± 2.4% vs. 58.4 ± 2.4%) and cartilage glycosaminoglycan content (499.0 ± 101.9 μg/mg dry weight vs. 155.0 ± 26.3 μg/mg dry weight) and were more effective at shifting OA synovial macrophage M1:M2 ratio (1.3:1 vs. 5:1), respectively. Taken together, the coculture model mimics salient features of OA, including macrophage-mediated cartilage destruction that was effectively abrogated by hAMSCs but not hADSCs. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants.

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    Timo Schomann

    Full Text Available Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs are promising candidates for this type of therapy, because they (1 have migratory properties, enabling migration after transplantation, (2 can differentiate into sensory neurons and glial cells, and (3 can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP. Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.

  16. A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.

    Science.gov (United States)

    Babini, Gabriele; Morini, Jacopo; Barbieri, Sofia; Baiocco, Giorgio; Ivaldi, Giovanni Battista; Liotta, Marco; Tabarelli de Fatis, Paola; Ottolenghi, Andrea

    2018-01-30

    The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

  17. PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

    Directory of Open Access Journals (Sweden)

    Ross Ferguson

    Full Text Available Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

  18. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Directory of Open Access Journals (Sweden)

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  19. Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel.

    Science.gov (United States)

    Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

    2016-12-01

    Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.

    Science.gov (United States)

    Fischer, J; Dickhut, A; Rickert, M; Richter, W

    2010-09-01

    The use of bone marrow-derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs. MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice. The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium. HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

  1. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    International Nuclear Information System (INIS)

    Ghislin, Stephanie; Obino, Dorian; Middendorp, Sandrine; Boggetto, Nicole; Alcaide-Loridan, Catherine; Deshayes, Frederique

    2012-01-01

    Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration

  2. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

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    Ghislin Stephanie

    2012-10-01

    Full Text Available Abstract Background Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18 expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. Methods A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. Results We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Conclusion Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

  3. The fabrication of double layer tubular vascular tissue engineering scaffold via coaxial electrospinning and its 3D cell coculture.

    Science.gov (United States)

    Ye, Lin; Cao, Jie; Chen, Lamei; Geng, Xue; Zhang, Ai-Ying; Guo, Lian-Rui; Gu, Yong-Quan; Feng, Zeng-Guo

    2015-12-01

    A continuous electrospinning technique was applied to fabricate double layer tubular tissue engineering vascular graft (TEVG) scaffold. The luminal layer was made from poly(ɛ-caprolac-tone)(PCL) ultrafine fibers via common single axial electrospinning followed by the outer layer of core-shell structured nanofibers via coaxial electrospinning. For preparing the outer layernano-fibers, the PCL was electrospun into the shell and both bovine serum albumin (BSA) and tetrapeptide val-gal-pro-gly (VAPG) were encapsulated into the core. The core-shell structure in the outer layer fibers was observed by transmission electron microscope (TEM). The in vitro release tests exhibited the sustainable release behavior of BSA and VAPG so that they provided a better cell growth environment in the interior of tubular scaffold wall. The in vitro culture of smooth muscle cells (SMCs) demonstrated their potential to penetrate into the scaffold wall for the 3D cell culture. Subsequently, 3D cell coculture was conducted. First, SMCs were seeded on the luminal surface of the scaffold and cultured for 5 days, and then endothelial cells (ECs) were also seeded on the luminal surface and cocultured with SMCs for another 2 days. After stained with antibodies, 3D cell distribution on the scaffold was revealed by confocal laser scanning microscopy (CLSM) where ECs were mainly located on the luminal surface whereas SMCs penetrated into the surface and distributed inside the scaffold wall. This double layer tubular scaffold with 3D cell distribution showed the promise to develop it into a novel TEVG for clinical trials in the near future. © 2015 Wiley Periodicals, Inc.

  4. Promotion of Vascular Morphogenesis of Endothelial Cells Co-Cultured with Human Adipose-Derived Mesenchymal Stem Cells Using Polycaprolactone/Gelatin Nanofibrous Scaffolds

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    Yun-Min Kook

    2018-02-01

    Full Text Available New blood vessel formation is essential for tissue regeneration to deliver oxygen and nutrients and to maintain tissue metabolism. In the field of tissue engineering, in vitro fabrication of new artificial vessels has been a longstanding challenge. Here we developed a technique to reconstruct a microvascular system using a polycaprolactone (PCL/gelatin nanofibrous structure and a co-culture system. Using a simple electrospinning process, we fabricated three-dimensional mesh scaffolds to support the sprouting of human umbilical vein endothelial cells (HUVECs along the electrospun nanofiber. The co-culture with adipose-derived mesenchymal stem cells (ADSCs supported greater sprouting of endothelial cells (ECs. In a two-dimensional culture system, angiogenic cell assembly produced more effective direct intercellular interactions and paracrine signaling from ADSCs to assist in the vascular formation of ECs, compared to the influence of growth factor. Although vascular endothelial growth factor and sphingosine-1-phosphate were present during the culture period, the presence of ADSCs was the most important factor for the construction of a cell-assembled structure in the two-dimensional culture system. On the contrary, HUVECs co-cultured on PCL/gelatin nanofiber scaffolds produced mature and functional microvessel and luminal structures with a greater expression of vascular markers, including platelet endothelial cell adhesion molecule-1 and podocalyxin. Furthermore, both angiogenic factors and cellular interactions with ADSCs through direct contact and paracrine molecules contributed to the formation of enhanced engineered blood vessel structures. It is expected that the co-culture system of HUVECs and ADSCs on bioengineered PCL/gelatin nanofibrous scaffolds will promote robust and functional microvessel structures and will be valuable for the regeneration of tissue with restored blood vessels.

  5. The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

    Science.gov (United States)

    Metzger, Wolfgang; Sossong, Daniela; Bächle, Annick; Pütz, Norbert; Wennemuth, Gunther; Pohlemann, Tim; Oberringer, Martin

    2011-09-01

    The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

  6. Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

    Science.gov (United States)

    VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

    2014-01-01

    The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

  7. Chondrocytes co-cultured with Stromal Vascular Fraction of adipose tissue present more intense chondrogenic characteristics than with Adipose Stem Cells

    NARCIS (Netherlands)

    Wu, Ling; Prins, H.J.; Leijten, Jeroen Christianus Hermanus; Helder, M.; Evseenko, D.; Moroni, L; van Blitterswijk, Clemens; Lin, Y.; Karperien, Hermanus Bernardus Johannes

    2016-01-01

    Partly replacement of chondrocytes by stem cells has been proposed to improve the performance of autologous chondrocytes implantation (ACI). Our previous studies showed that the increased cartilage production in pellet co-cultures of chondrocytes and mesenchymal stem cells (MSCs) is due to a trophic

  8. Effects of Alpha Particle and Proton Beam Irradiation as Putative Cross-Talk between A549 Cancer Cells and the Endothelial Cells in a Co-Culture System

    International Nuclear Information System (INIS)

    Riquier, Hélène; Abel, Denis; Wera, Anne-Catherine; Heuskin, Anne-Catherine; Genard, Géraldine; Lucas, Stéphane; Michiels, Carine

    2015-01-01

    Background: High-LET ion irradiation is being more and more often used to control tumors in patients. Given that tumors are now considered as complex organs composed of multiple cell types that can influence radiosensitivity, we investigated the effects of proton and alpha particle irradiation on the possible radioprotective cross-talk between cancer and endothelial cells. Materials and Methods: We designed new irradiation chambers that allow co-culture study of cells irradiated with a particle beam. A549 lung carcinoma cells and endothelial cells (EC) were exposed to 1.5 Gy of proton beam or 1 and 2 Gy of alpha particles. Cell responses were studied by clonogenic assays and cell cycle was analyzed by flow cytometry. Gene expression studies were performed using Taqman low density array and by RT-qPCR. Results: A549 cells and EC displayed similar survival fraction and they had similar cell cycle distribution when irradiated alone or in co-culture. Both types of irradiation induced the overexpression of genes involved in cell growth, inflammation and angiogenesis. Conclusions: We set up new irradiation chamber in which two cell types were irradiated together with a particle beam. We could not show that tumor cells and endothelial cells were able to protect each other from particle irradiation. Gene expression changes were observed after particle irradiation that could suggest a possible radioprotective inter-cellular communication between the two cell types but further investigations are needed to confirm these results

  9. Effects of Alpha Particle and Proton Beam Irradiation as Putative Cross-Talk between A549 Cancer Cells and the Endothelial Cells in a Co-Culture System

    Energy Technology Data Exchange (ETDEWEB)

    Riquier, Hélène; Abel, Denis [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium); Wera, Anne-Catherine; Heuskin, Anne-Catherine [LARN-PMR, NARILIS, University of Namur, Namur 5000 (Belgium); Genard, Géraldine [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium); Lucas, Stéphane [LARN-PMR, NARILIS, University of Namur, Namur 5000 (Belgium); Michiels, Carine, E-mail: carine.michiels@unamur.be [URBC-NARILIS, University of Namur, 61 rue de Bruxelles, Namur 5000 (Belgium)

    2015-03-18

    Background: High-LET ion irradiation is being more and more often used to control tumors in patients. Given that tumors are now considered as complex organs composed of multiple cell types that can influence radiosensitivity, we investigated the effects of proton and alpha particle irradiation on the possible radioprotective cross-talk between cancer and endothelial cells. Materials and Methods: We designed new irradiation chambers that allow co-culture study of cells irradiated with a particle beam. A549 lung carcinoma cells and endothelial cells (EC) were exposed to 1.5 Gy of proton beam or 1 and 2 Gy of alpha particles. Cell responses were studied by clonogenic assays and cell cycle was analyzed by flow cytometry. Gene expression studies were performed using Taqman low density array and by RT-qPCR. Results: A549 cells and EC displayed similar survival fraction and they had similar cell cycle distribution when irradiated alone or in co-culture. Both types of irradiation induced the overexpression of genes involved in cell growth, inflammation and angiogenesis. Conclusions: We set up new irradiation chamber in which two cell types were irradiated together with a particle beam. We could not show that tumor cells and endothelial cells were able to protect each other from particle irradiation. Gene expression changes were observed after particle irradiation that could suggest a possible radioprotective inter-cellular communication between the two cell types but further investigations are needed to confirm these results.

  10. Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2/HT29-MTX co-cultures.

    Science.gov (United States)

    Akbari, Ali; Lavasanifar, Afsaneh; Wu, Jianping

    2017-12-01

    The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery. The long-term objective of

  11. Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method.

    Science.gov (United States)

    Eberlein, B; Baur, A; Neundorfer, M; Jahn, G

    1991-05-01

    Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

  12. A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells

    Directory of Open Access Journals (Sweden)

    Fanny Ehret

    2015-11-01

    Full Text Available The niche concept of stem cell biology proposes a functional unit between the precursor cells and their local microenvironment, to which several cell types might contribute by cell–cell contacts, extracellular matrix, and humoral factors. We here established three co-culture models (with cell types separated by membrane for both adherent monolayers and neurospheres to address the potential influence of different niche cell types in the neurogenic zone of the adult hippocampus of mice. Astrocytes and endothelial cells enhanced precursor cell proliferation and neurosphere formation. Endothelial factors also led to a prolonged increase in proliferation after growth factor withdrawal, which otherwise induces differentiation. All niche cell types enhanced cell survival in monolayer cultures, endothelial cells also stimulated neuronal differentiation. A parallel trend elicited by astrocytes did not reach conventional statistical significance. Pericytes had variable effects here. We did not observe changes in differentiation in neurosphere co-cultures. In summary, our data indicate that in precursor cell culture protocols survival could be improved by adding as yet unknown factors physiologically contributed by astrocytes and endothelial cells. Our findings also underscore the complexity of the niche and the differential impact of factors from the different sources on distinct aspects of neuronal development. With the help of the models presented here, identification of these factors and their specific biological activity can now be initiated.

  13. Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenjie [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Zhang, Xiaomei, E-mail: zhangxm667@163.com [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Lu, Hong [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Matsukura, Makoto [Laboratory of Clinical Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082 (Japan); Zhao, Jien; Shinohara, Makoto [Ashikita Institution for Developmental Disabilities, 2813 Oaza Ashikita, Ashikita-machi, Ashikita, Kumamoto 869-5461 (Japan)

    2013-05-10

    Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

  14. A co-culture microtunnel technique demonstrating a significant contribution of unmyelinated Schwann cells to the acceleration of axonal conduction in Schwann cell-regulated peripheral nerve development.

    Science.gov (United States)

    Sakai, Koji; Shimba, Kenta; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2017-08-14

    Schwann cells (SCs) contribute to the regulation of axonal conduction in a myelin-dependent and -independent manner. However, due to the lack of investigative techniques that are able to record axonal conduction under conditions that control the proliferation of specific SC types, little is known about the extent to which myelinated SCs (mSCs) and unmyelinated SCs (umSCs) modulate axonal conduction. In this study, a microtunnel-electrode approach was applied to a neuron/SC co-culture technique. Rat dorsal root ganglion neurons and SCs were co-cultured in a microtunnel-electrode device, which enabled recording of the conduction delay in multiple axons passing through microtunnels. Despite the absence of nuclei in the microtunnel when SCs were eliminated, cultured cells were densely packed and expressed S100 beta (an SC marker) at a rate of 96% in neuron/SC co-culture, indicating that SCs migrated into the microtunnel. In addition, supplementation with ascorbic acid after 6 days in vitro (DIV) successfully induced myelination from 22 DIV. Activity recording experiments indicated that the conduction delay decreased with culture length from 17 DIV in the neuron/SC co-culture but not in neuron monoculture. Interestingly, the SC-modulated shortening of conduction delay was attenuated at 17 DIV and 22 DIV by supplementing the culture medium with ascorbic acid and, at the same time, suppressing SC proliferation, suggesting that immature umSCs increased axonal conduction velocity in a cell density-dependent manner before the onset of myelination. These results suggest that this method is an effective tool for investigating the contributions of mSCs or umSCs to the regulation of axonal conduction.

  15. The angiogenic behaviors of human umbilical vein endothelial cells (HUVEC) in co-culture with osteoblast-like cells (MG-63) on different titanium surfaces.

    Science.gov (United States)

    Shi, Bin; Andrukhov, Oleh; Berner, Simon; Schedle, Andreas; Rausch-Fan, Xiaohui

    2014-08-01

    Interaction between osteogenesis and angiogenesis plays an important role in implant osseointegration. In the present study we investigated the influence of titanium surface properties on the angiogenic behaviors of endothelial cells grown in direct contact co-culture with osteoblasts. Human umbilical vein endothelial cells (HUVECs) and osteoblast-like cells (MG-63 cells) were grown in direct co-culture on the following titanium surfaces: acid-etched (A), hydrophilic A (modA), coarse-gritblasted and acid-etched (SLA) and hydrophilic SLA (SLActive). Cell proliferation was evaluated by cell counting combined with flow cytometry. The expression of von Willebrand Factor (vWF), thrombomodulin (TM), endothelial cell protein C receptor (EPCR), E-Selectin, as well as vascular endothelial growth factor (VEGF) receptors Flt-1 and KDR in HUVECs and VEGF in MG-63 were measured by qPCR. The dynamic behavior of endothelial cells was recorded by time-lapse microscopy. Proliferation of HUVECs was highest on A, followed by SLA, modA and SLActive surfaces. The expression of vWF, TM, EPCR, E-Selectin and Flt-1 in HUVECs was significantly higher on A than on all other surfaces. The expression of KDR in HUVECs grown on A surface was below detection limit. VEGF expression in MG-63 cells was significantly higher on SLActive vs SLA and modA vs A surfaces. Time-lapse microscopy revealed that HUVECs moved quickest and formed cell clusters earlier on A surface, followed by SLA, modA and SLActive surface. In co-culture conditions, proliferation and expression of angiogenesis associated genes in HUVECs are promoted by smooth hydrophobic Ti surface, which is in contrast to previous mono-culture studies. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  16. Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal.

    Science.gov (United States)

    Chen, Yu-Chih; Cheng, Yu-Heng; Kim, Hong Sun; Ingram, Patrick N; Nor, Jacques E; Yoon, Euisik

    2014-08-21

    Cancer-stromal cell interactions are a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, have limitations in three aspects: 1) limited control of the cell microenvironment; 2) inability to study cell behavior in a single-cell manner; and 3) have difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is for improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells while maintaining nutrient renewal. In this work, two-phase flow was used as a simple isolation method, separating the microenvironment of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect the cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows area reduction of vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between the cell culture layer and the media exchange layer. The selectivity of the semi-permeable membrane results in the retention of the signaling proteins within the chamber while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk between cells in other chambers. Utilizing these innovations, we co-cultured UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to simulate tumor proliferation enhancement in the vascular endothelial niche.

  17. MCF10A and MDA-MB-231 human breast basal epithelial cell co-culture in silicon micro-arrays.

    Science.gov (United States)

    Nikkhah, Mehdi; Strobl, Jeannine S; Schmelz, Eva M; Roberts, Paul C; Zhou, Hui; Agah, Masoud

    2011-10-01

    We developed istotropically etched silicon chip micro-arrays for co-culture of metastatic human breast cancer (MDA-MB-231) and non-tumorigenic human breast (MCF10A) cells. The micro-arrays were fabricated using a single-mask, single-etch step process. Each chip contained a 16×16 array of cavities 140 μm wide by 60 μm deep separated by planar silicon surfaces. Cells occupied 97-100% of the etched cavities. The cavities were enriched three-fold in MDA-MB-231 cells relative to the seeding ratio of, MDA-MB-231(1): MCF10A(10) cells. Micro co-cultures comprised of both MCF10A and MDA-MB-231 cells formed in 26% of cavities and contained 2-10 cells per cavity. Heterotropic cell interactions were seen in co-culture, and sites of these interactions were enriched with vinculin spikes. A selective morphological response to the histone deacetylase inhibitor (HDI), SAHA (suberoylanilide hydroxamic acid), occurred in MDA-MB-231 cells which was quantified by significant increases in cell length and cell area on flat surfaces and in the number of stretched cells inside the etched cavities. The morphology of MCF10A cells was unaltered in response to SAHA. Real time imaging showed the formation of highly dynamic and randomly orienting cytoplasmic extensions in MDA-MB-231 cells 1h after adding SAHA; this is the first report of a rapid, morphological response in breast tumor cells to a histone deacetylase inhibitor. The findings demonstrate the utility of etched silicon micro-arrays for the propagation of human breast cell co-cultures and the application of HDI as a potential marker to distinguish metastatic breast cancer cells in a background of normal breast cell types. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Protective role of klotho protein on epithelial cells upon co-culture with activated or senescent monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Mytych, Jennifer, E-mail: jennifermytych@gmail.com [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Wos, Izabela; Solek, Przemyslaw; Koziorowski, Marek [Institute of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland); Centre of Applied Biotechnology and Basic Sciences, University of Rzeszow, Werynia 502, 36-100 Kolbuszowa (Poland)

    2017-01-15

    Monocytes ensure proper functioning and maintenance of epithelial cells, while good condition of monocytes is a key factor of these interactions. Although, it was shown that in some circumstances, a population of altered monocytes may appear, there is no data regarding their effect on epithelial cells. In this study, using direct co-culture model with LPS-activated and Dox-induced senescent THP-1 monocytes, we reported for the first time ROS-induced DNA damage, reduced metabolic activity, proliferation inhibition and cell cycle arrest followed by p16-, p21- and p27-mediated DNA damage response pathways activation, premature senescence and apoptosis induction in HeLa cells. Also, we show that klotho protein possessing anti-aging and anti-inflammatory characteristics reduced cytotoxic and genotoxic events by inhibition of insulin/IGF-IR and downregulation of TRF1 and TRF2 proteins. Therefore, klotho protein could be considered as a protective factor against changes caused by altered monocytes in epithelial cells. - Highlights: • Activated and senescent THP-1 monocytes induced cyto- and genotoxicity in HeLa cells. • Altered monocytes provoked oxidative and nitrosative stress-induced DNA damage. • DNA damage activated DDR pathways and lead to premature senescence and apoptosis. • Klotho reduced ROS/RNS-mediated toxicity through insulin/IGF-IR pathway inhibition. • Klotho protects HeLa cells from cyto- and genotoxicity induced by altered monocytes.

  19. Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode

    Science.gov (United States)

    Berger, Emmanuelle; Nassra, Merian; Atgié, Claude; Plaisancié, Pascale; Géloën, Alain

    2017-01-01

    Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells. PMID:28726765

  20. Biological responses of T cells encapsulated with polyelectrolyte-coated gold nanorods and their cellular activities in a co-culture system

    Science.gov (United States)

    Wattanakull, Porntida; Killingsworth, Murray C.; Pissuwan, Dakrong

    2017-11-01

    Currently, human T cell therapy is of considerable scientific interest. In addition, cell encapsulation has become an attractive approach in biomedical applications. Here, we propose an innovative technique of single-cell encapsulation of human T cells using polyelectrolytes combined with gold nanorods. We have demonstrated encapsulation of human Jurkat T cells with poly(sodium 4-styrenesulfonate) (PSS)-coated gold nanorods (PSS-GNRs). Other forms of encapsulation, using polyelectrolytes without GNRs, were also performed. After Jurkat T cells were encapsulated with poly(allylamine hydrochloride) (PAH) and/or PSS-GNRs or PSS, most cells survived and could proliferate. Jurkat T cells encapsulated with a double layer of PSS-GNR/PAH (PSS-GNR/PAH@Jurkat) showed the highest rate of cell proliferation when compared to 24-h encapsulated cells. With the exception of IL-6, no significant induction of inflammatory cytokines (IL-2, IL-1β, and TNF-α) was observed. Interestingly, when encapsulated cells were co-cultured with THP-1 macrophages, co-cultures exhibited TNF-α production enhancement. However, the co-culture of THP-1 macrophage and PSS-GNR/PAH@Jurkat or PSS/PAH@Jurkat did not enhance TNF-α production. No significant inductions of IL-2, IL-1β, and IL-6 were detected. These data provide promising results, demonstrating the potential use of encapsulated PSS-GNR/PAH@Jurkat to provide a more inert T cell population for immunotherapy application and other biomedical applications.

  1. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    Energy Technology Data Exchange (ETDEWEB)

    Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  2. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    International Nuclear Information System (INIS)

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-01-01

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes

  3. Single-cell qPCR facilitates the optimization of hematopoietic differentiation in hPSCs/OP9 coculture system.

    Science.gov (United States)

    Chen, Haide; Jiang, Mengmeng; Xiao, Lei; Huang, He

    2018-03-15

    Human pluripotent stem cells (hPSCs)/OP9 coculture system is a widely used hematopoietic differentiation approach. The limited understanding of this process leads to its low efficiency. Thus, we used single-cell qPCR to reveal the gene expression profiles of individual CD34+ cells from different stages of differentiation. According to the dynamic gene expression of hematopoietic transcription factors, we overexpressed specific hematopoietic transcription factors (Gata2, Lmo2, Etv2, ERG, and SCL) at an early stage of hematopoietic differentiation. After overexpression, we generated more CD34+ cells with normal expression level of CD43 and CD31, which are used to define various hematopoietic progenitors. Furthermore, these CD34+ cells possessed normal differentiation potency in colony-forming unit assays and normal gene expression profiles. In this study, we demonstrated that single-cell qPCR can provide guidance for optimization of hematopoietic differentiation and transient overexpression of selected hematopoietic transcription factors can enhance hematopoietic differentiation.

  4. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells.

    Science.gov (United States)

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (Pculture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

  5. Proliferating fibroblasts and HeLa cells co-cultured in vitro reciprocally influence growth patterns, protein expression, chromatin features and cell survival.

    Science.gov (United States)

    Delinasios, John G; Angeli, Flora; Koumakis, George; Kumar, Shant; Kang, Wen-Hui; Sica, Gigliola; Iacopino, Fortunata; Lama, Gina; Lamprecht, Sergio; Sigal-Batikoff, Ina; Tsangaris, George T; Farfarelos, Christos D; Farfarelos, Maria C; Vairaktaris, Eleftherios; Vassiliou, Stavros; Delinasios, George J

    2015-04-01

    to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro. Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment. Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed

  6. The impact of simulated microgravity on purinergic signaling in an endothelial and smooth muscle cell co-culture model

    Science.gov (United States)

    Zhang, Yu; Hemmersbach, Ruth; Lau, Patrick; Pansky, Andreas; Kassack, Matthias; Tobiasch, Edda

    Astronauts suffer from cardiovascular deconditioning when they are exposed to microgravity conditions during space missions. Thus, current research focuses on the identification of the underlying mechanism also with respect to therapy and countermeasures. Endothelial cells (ECs) and smooth muscle cells (SMCs) play a key role in a variety of vascular functions. Gene expression, cytoskeleton morphology and apoptosis in both, ECs and SMCs, have shown alterations under simulated and real microgravity condition. However, all these data were observed during single culturing of either ECs or SMCs under microgravity conditions, which is different from the in vivo situation. Purinergic 2 (P2) receptors bind extracellular nucleotides and can regulate the vascular tone and vascular cell proliferation, migration and apoptosis. In this study primary ECs and SMCs were obtained from bovine aorta and characterized using specific markers. Here we show for the first time that the P2-receptor expressions pattern in ECs and in SMCs is altered after 24h in simulated microgravity. Specific receptors are down- or up-regulated on the gene and protein level. In addition the supernatant of ECs during culture was used as conditioned medium for SMCs and vice visa to investigate the influence of either cell type on the other. ECs and SMCs secret cytokines which induce pathogenic proliferation and an altered migration behavior under simulated microgravity conditions. Interestingly, co-culturing with condition medium could compensate this change. In detail, P2X7 was down-regulated in ECs after 24h clinorotation but recovered to the 1 g level when cultured with conditioned medium from SMCs collected under normal gravity. In conclusion, our data indicate that the paracrine effect between ECs and SMCs is an important regulator of cell behavior, also under altered gravity conditions. P2-receptor gene and protein expression were altered during microgravity. Since several P2-receptor artificial

  7. Immunoregulatory effects of human dental pulp-derived stem cells on T cells: comparison of transwell co-culture and mixed lymphocyte reaction systems.

    Science.gov (United States)

    Demircan, Pinar Cetinalp; Sariboyaci, Ayla Eker; Unal, Zehra Seda; Gacar, Gulcin; Subasi, Cansu; Karaoz, Erdal

    2011-11-01

    BACKGROUND AIMS. Studies performed using human and animal models have indicated the immunoregulatory capability of mesenchymal stromal cells in several lineages. We investigated whether human dental pulp-derived stem cells (hDP-SC) have regulatory effects on phytohemagglutinin (PHA)-activated CD3(+) T cells. We aimed to define the regulatory mechanisms associated with hDP-SC that occur in mixed lymphocyte reaction (MLR) and transwell systems with PHA-CD3(+) T cells and hDP-SC at a ratio of 1:1. METHODS. Proliferation, apoptosis and pro- and anti-inflammatory cytokines of PHA-CD3(+)T cells, the expression of Regulatory T cells (Treg) markers and some regulatory factors related to hDP-SC, were studied in Both transwell and MLR are co-cultures systems. RESULTS. Anti-proliferative and apoptotic effects of hDP-SC were determined in co-culture systems. Elevated expression levels of human leukocyte antigen (HLA)-G, hepatocyte growth factor (HGF)-β1, intracellular adhesion molecule (ICAM-1)-1, interleukin (IL)-6, IL-10, transforming growth factor (TGF)-β1, vascular adhesion molecule (VCAM)-1 and vascular endothelial growth factor (VEGF) by hDP-SC were detected in the co-culture systems. We observed decreased expression levels of pro-inflammatory cytokines [interferon (IFN)-γ, IL-2, IL-6 receptor (R), IL-12, Interleukin-17A (IL-17A), tumor necrosis factor (TNF)-α] and increased expression levels of anti-inflammatory cytokine [inducible protein (IP)-10] from PHA-CD3(+) T cells in the transwell system. Expression of Treg (CD4(+) CD25(+) Foxp3(+)) markers was significantly induced by hDP-SC in both co-culture systems. We observed apoptosis of PHA-CD3(+) T cells with 24 h using time-lapse camera photographs and active caspase labeling; it is likely that paracrine soluble factors and molecular signals secreted by hDP-SC led this apoptosis. CONCLUSIONS. We suggest that hDP-SC have potent immunoregulatory functions because of their soluble factors and cytokines via paracrine

  8. Successful use of oviduct epithelial cell coculture for in vitro production of viable red deer (Cervus elaphus) embryos.

    Science.gov (United States)

    Locatelli, Y; Cognié, Y; Vallet, J C; Baril, G; Verdier, M; Poulin, N; Legendre, X; Mermillod, P

    2005-11-01

    Techniques for in vitro production (IVP) of viable embryos have been thoroughly developed in several domestic species in view to improve breeding efficiency. When applied to wild life, these techniques may also help the maintenance of biodiversity through amplification of sparse animals offspring and facilitation of genetic material exchange. During the successive steps of IVP, i.e. oocyte in vitro maturation (IVM), fertilization (IVF) and early embryo development (IVD) to the blastocyst stage, gametes and embryos are faced with unusual environment, including oxidative stress, known to be detrimental to their survival. In the present study, starting from methods developed in domestic species, we have adapted IVP to produce viable red deer embryos. In a first experiment, cumulus cells were removed from in vitro matured oocytes either before or after IVF. The presence of cumulus cells during IVF did not affect final cleavage or development rates. In a second experiment, in vitro matured oocytes were fertilized in the presence of cumulus cells and cultured in SOFaaBSA medium alone or in the presence of ovine oviduct epithelial cell (oOEC) monolayer. Whereas, oviduct cells did not improve the cleavage rate, they significantly increased the rate of embryos reaching the blastocyst stage (from 3 to 25% of total oocytes). Ten blastocysts from oOEC coculture were transferred after freezing and thawing to five recipient hinds and gave rise to three pregnancies. The three pregnant hinds gave birth to three live and normal calves.

  9. Isotropic Versus Bipolar Functionalized Biomimetic Artificial Basement Membranes and Their Evaluation in Long-Term Human Cell Co-Culture.

    Science.gov (United States)

    Rossi, Angela; Wistlich, Laura; Heffels, Karl-Heinz; Walles, Heike; Groll, Jürgen

    2016-08-01

    In addition to dividing tissues into compartments, basement membranes are crucial as cell substrates and to regulate cellular behavior. The development of artificial basement membranes is indispensable for the ultimate formation of functional engineered tissues; however, pose a challenge due to their complex structure. Herein, biodegradable electrospun polyester meshes are presented, exhibiting isotropic or bipolar bioactivation as a biomimetic and biofunctional model of the natural basement membrane. In a one-step preparation process, reactive star-shaped prepolymer additives, which generate a hydrophilic fiber surface, are electrospun with cell-adhesion-mediating peptides, derived from major components of the basement membrane. Human skin cells adhere to the functionalized meshes, and long-term co-culture experiments confirm that the artificial basement membranes recapitulate and preserve tissue specific functions. Several layers of immortalized human keratinocytes grow on the membranes, differentiating toward the surface and expressing typical epithelial markers. Fibroblasts migrate into the reticular lamina mimicking part of the mesh. Both cells types begin to produce extracellular matrix proteins and to remodel the initial membrane. It is shown at the example of skin that the artificial basement membrane design provokes biomimetic responses of different cell types and can thus be used as basis for the future development of basement membrane containing tissues. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

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    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  11. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line.

    Science.gov (United States)

    Desai, Nina; Ludgin, Jennifer; Goldberg, Jeffrey; Falcone, Tommaso

    2013-06-01

    Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2-3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

  12. Effects of low oxygen tension on gene profile of soluble growth factors in co-cultured adipose-derived stromal cells and chondrocytes.

    Science.gov (United States)

    Shi, Sirong; Xie, Jing; Zhong, Juan; Lin, Shiyu; Zhang, Tao; Sun, Ke; Fu, Na; Shao, Xiaoru; Lin, Yunfeng

    2016-06-01

    Moving towards development of optimized cartilage regeneration with adipose-derived stromal cells (ASCs), the focus of this study was on investigating the influence of hypoxia on soluble factors secreted by ASCs and chondrocytes after crosstalk. We established direct contact co-culture and non-contact co-culture systems by using red or green fluorescent protein (R/GFP)-labelled mice and SD rats respectively. Gene variation of growth factors of the two cell types, in both hypoxic and normoxic conditions, were screened using semi-quantitative polymerase chain reaction (PCR). Co-culture with ASCs and chondrocytes under hypoxia was shown to successfully induce or enhance ASC to chondrogenic differentiation. To be specific, chondrogenic maker genes: AGC, COL II and SOX9 were remarkably enhanced in both ASCs and chondrocytes after crosstalk under low oxygen tension. Subsequently, screening growth factors in ASCs and chondrocytes under hypoxia showed that HIF-1α, VEGF-A/B, BMP-2/-4/-6, FGF-2 and IGF-1 were significantly increased, but not TGF-β1. These results revealed that both hypoxia and co-culture systems can notably enhance chondrogenesis of ASCs as well as increase proliferation of ASCs and chondrocytes. © 2016 John Wiley & Sons Ltd.

  13. Production of plant cell wall degrading enzymes by monoculture and co-culture of Aspergillus niger and Aspergillus terreus under SSF of banana peels

    Directory of Open Access Journals (Sweden)

    Shazia Rehman

    2014-12-01

    Full Text Available Filamentous fungi are considered to be the most important group of microorganisms for the production of plant cell wall degrading enzymes (CWDE, in solid state fermentations. In this study, two fungal strains Aspergillus niger MS23 and Aspergillus terreus MS105 were screened for plant CWDE such as amylase, pectinase, xylanase and cellulases (β-glucosidase, endoglucanase and filterpaperase using a novel substrate, Banana Peels (BP for SSF process. This is the first study, to the best of our knowledge, to use BP as SSF substrate for plant CWDE production by co-culture of fungal strains. The titers of pectinase were significantly improved in co-culture compared to mono-culture. Furthermore, the enzyme preparations obtained from monoculture and co-culture were used to study the hydrolysis of BP along with some crude and purified substrates. It was observed that the enzymatic hydrolysis of different crude and purified substrates accomplished after 26 h of incubation, where pectin was maximally hydrolyzed by the enzyme preparations of mono and co-culture. Along with purified substrates, crude materials were also proved to be efficiently degraded by the cocktail of the CWDE. These results demonstrated that banana peels may be a potential substrate in solid-state fermentation for the production of plant cell wall degrading enzymes to be used for improving various biotechnological and industrial processes.

  14. A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

    Science.gov (United States)

    McConda, David B; Karnes, Jonathan M; Hamza, Therwa; Lindsey, Brock A

    2016-07-01

    Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.

  15. In Vitro Large Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    2013-01-01

    Full Text Available In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs. HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive β-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

  16. CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

    Science.gov (United States)

    Jia, Xiaoyi; Wei, Fang; Sun, Xiaojing; Chang, Yan; Xu, Shu; Yang, Xuezhi; Wang, Chun; Wei, Wei

    2016-08-02

    Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability

  17. Effects of cigarette smoke extract on A549 cells and human lung fibroblasts treated with transforming growth factor-beta1 in a coculture system.

    Science.gov (United States)

    Liu, Yin; Gao, Wei; Zhang, Deping

    2010-09-01

    Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the mechanism of the association remains unknown. The aim of this study was to investigate the effects of cigarette smoke extract (CSE) on A549 cells and human lung fibroblasts treated with transforming growth factor-beta1. A transwell two-chamber coculture system was used to study the proliferation, differentiation, morphologic changes and soluble factors production of A549 cells and myofibroblasts. Low concentrations of CSE promoted myofibroblasts proliferation; however, high concentrations of CSE inhibited their proliferation. Low concentrations of CSE also markedly increased extracellular secretion of hydrogen peroxide, inhibited proliferation, induced apoptosis and produced epithelial-mesenchymal transition (EMT) in cocultured A549 cells. This cigarette smoke-induced A549 cells EMT may become a new pathophysiological concept in the development of IPF. CSE possibly takes part in the development and progress of IPF by increasing oxidative stress.

  18. Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

    Science.gov (United States)

    Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

    2017-08-15

    Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

  19. Functional innervation of human induced pluripotent stem cell-derived cardiomyocytes by co-culture with sympathetic neurons developed using a microtunnel technique.

    Science.gov (United States)

    Sakai, Koji; Shimba, Kenta; Ishizuka, Kazuma; Yang, Zhuonan; Oiwa, Kosuke; Takeuchi, Akimasa; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2017-12-09

    Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed an MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment.

    Science.gov (United States)

    Chen, Miaojiao; Zhang, Jingjing; Hu, Fang; Liu, Shiping; Zhou, Zhiguang

    2015-11-01

    Accumulating evidence suggests an association between diabetes and cancer. Inflammation is a key event that underlies the pathological processes of the two diseases. Metformin displays anti-cancer effects, but the mechanism is not completely clear. This study investigated whether metformin regulated the microenvironment of macrophage polarization to affect the characteristics of HepG2 cells and the possible role of the Notch-signalling pathway. RAW264.7 macrophages were cultured alone or co-cultured with HepG2 cells and treated with metformin. We analysed classical (M1) and alternative (M2) gene expression in RAW264.7 cells using quantitative real-time polymerase chain reaction. Changes in mRNA and protein expressions of Notch signalling in both cell types were also detected using quantitative real-time polymerase chain reaction and Western-blotting analyses. The proliferation, apoptosis and migration of HepG2 cells were detected using Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega Corporation, Fitchburg, WI, USA), Annexin V-FITC/PI (7SeaPharmTech, Shanghai, China) and the cell scratch assay, respectively. Metformin induced single-cultured RAW264.7 macrophages with an M2 phenotype but attenuated the M2 macrophage differentiation and inhibited monocyte chemoattractant protein-1 (MCP-1) secretion in a co-culture system. The co-cultured group of metformin pretreatment activated Notch signalling in macrophages but repressed it inHepG2 cells. Co-culture also promoted the proliferation and migration of HepG2 cells. However, along with the enhanced apoptosis, the proliferation and the migration of HepG2 cells were remarkably inhibited in another co-culture system with metformin pretreatment. Metformin can skew RAW264.7 macrophages toward different phenotypes according to changes in the microenvironment, which may affect the inflammatory conditions mediated by macrophages, induce apoptosis and inhibit the proliferation and migration of HepG2

  1. Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells.

    Science.gov (United States)

    Wright, Karina T; Uchida, Kenzo; Bara, Jennifer J; Roberts, Sally; El Masri, Wagih; Johnson, William E B

    2014-08-01

    Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural

  2. A study of a three-dimensional PLGA sponge containing natural polymers co-cultured with endothelial and mesenchymal stem cells as a tissue engineering scaffold.

    Science.gov (United States)

    Shim, Jung Bo; Ankeny, Randall F; Kim, Hyeongseok; Nerem, Robert M; Khang, Gilson

    2014-08-01

    The interaction between vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in a complex hemodynamic and mechanical environment plays an important role in the control of blood vessel growth and function. Despite the importance of VSMCs, substitutes are needed for vascular therapies. A potential VSMC substitute is human adult bone marrow derived mesenchymal stem cells (hMSCs). In this study, the effect of poly(lactic-co-glycolic acid) (PLGA) scaffolds containing three natural polymers (demineralized bone particles, silk, and small intestine submucosa) on the phenotype of MSCs and SMCs cultured with or without ECs was investigated. The study objective was to create a media equivalent for a tissue engineered blood vessel using PLGA, natural polymers, and MSCs co-cultured with ECs. The PLGA containing the natural polymers silk and SIS showed increased proliferation and cell adhesion. The presence of silk and DBP promoted a MSC phenotype change into a SMC-like phenotype at the mRNA level; however these differences at the protein level were not seen. Additionally, PLGA containing SIS did not induce SMC gene or protein upregulation. Finally, the effect of ECs in combination with the natural polymers was tested. When co-cultured with ECs, the mRNA of SMC specific markers in MSCs and SMCs were increased when compared to SMCs or MSCs alone. However, MSCs, when co-cultured with ECs on PLGA containing silk, exhibited significantly increased α-SMA and calponin expression when compared to PLGA only scaffolds. These results indicate that the natural polymer silk in combination with the co-culture of endothelial cells was most effective at increasing cell viability and inducing a SMC-like phenotype at the mRNA and protein level in MSCs.

  3. Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Valentina Paracchini

    2012-01-01

    Full Text Available Cystic fibrosis (CF is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

  4. Attack the tumor counterattack-c-FLIP expression in Jurkat-T-cells protects against apoptosis induced by coculture with SW620 colorectal adenocarcinoma cells.

    Science.gov (United States)

    Steiert, Andreas E; Sendler, Daniel; Burke, Willam F; Choi, Claudia Y; Reimers, Kerstin; Vogt, Peter M

    2012-07-01

    Cancer development relies on a variety of mechanisms that facilitate tumor growth despite the presence of a functioning immune system, employing different mechanisms to escape immune rejection. Tumors may eliminate tumor-infiltrating lymphocytes and suppress anti-tumor immune responses, a process called "tumor counterattack," based on activation-induced cell death via the FAS/FAS-ligand system. To overcome this tumor-cell survival strategy, we examined the hypothesis that the sensitivity of FAS mediated apoptosis of Jurkat-T-cells can be suppressed by FLIP transfection of Jurkat-T-cells. Jurkat-T-cells were transfected with the FLICE-inhibitory protein FLIP in order to bestow them with a resistance to FAS-receptor-mediated apoptosis. FLIP-transfected and non-transfected Jurkat-T-cells were grown in coincubation with SW620 cells and the rates of apoptosis measured via FACS-analysis of Annexin-V. First, the tumor-counterattack described in the literature was confirmed. About 20% of Jurkat-T-Cells underwent apoptosis in coculture with SW620 cells. After coincubation of SW620 cells with FLIP transfected Jurkat-T-cells the apoptotic rate was significant reduced at levels below 4%. Transfection of Jurkat-T-cells with FLIP reduces the sensitivity of Jurkat-T-cells to FAS-mediated apoptosis and may lead to an improved capability to antagonize the inherent tumor survival strategy of SW620 cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Collective motion of cells mediates segregation and pattern formation in co-cultures.

    Directory of Open Access Journals (Sweden)

    Elod Méhes

    Full Text Available Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion.

  6. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  7. A new cell line-based coculture model of the human air-blood barrier to evaluate the interaction with aerosolized drug carriers

    OpenAIRE

    Kletting, Stephanie

    2016-01-01

    Besides reducing animal testing, in vitro models allow for the pre-screening of new drug candidates in terms of safety and efficacy before they enter clinical trials. To date, models mimicking the deep lung show limitations such as cellular origin or lack of appropriate barrier properties. Therefore, the focus of this work was on the establishment of a robust and reproducible cell line-based coculture model that reflects the two major barrier structures present in the alveolar region, namely ...

  8. Fighting for territories: time-lapse analysis of dental pulp and dental follicle stem cells in co-culture reveals specific migratory capabilities

    Directory of Open Access Journals (Sweden)

    C Schiraldi

    2012-11-01

    Full Text Available Stem cell migration is a critical step during the repair of damaged tissues. In order to achieve appropriate cell-based therapies for tooth and periodontal ligament repair it is necessary first to understand the dynamics of tissue-specific stem cell populations such as dental pulp stem cells (DPSC and dental follicle stem cells (DFSC. Using time-lapse imaging, we analysed migratory and proliferative capabilities of these two human stem cell lines in vitro. When cultured alone, both DPSC and DFSC exhibited low and irregular migration profiles. In co-cultures, DFSC, but not DPSC, spectacularly increased their migration activity and velocity. DFSC rapidly surrounded the DPSC, thus resembling the in vivo developmental process, where follicle cells encircle both dental epithelium and pulp. Cell morphology was dependent on the culture conditions (mono-culture or co-culture and changed over time. Regulatory genes involved in dental cell migration and differentiation such as TWIST1, MSX1, RUNX2, SFRP1 and ADAM28, were also evaluated in co-cultures. MSX1 up-regulation indicates that DPSC and DFSC retain their odontogenic potential. However, DPSC lose their capacity to differentiate into odontoblasts in the presence of DFSC, as suggested by RUNX2 up-regulation and TWIST1 down-regulation. In contrast, the unchanged levels of SFRP1 expression suggest that DFSC retain their potential to form periodontal tissues even in the presence of DPSC. These findings demonstrate that stem cells behave differently according to their environment, retain their genetic memory, and compete with each other to acquire the appropriate territory. Understanding the mechanisms involved in stem cell migration may lead to new therapeutic approaches for tooth repair.

  9. , , , , , and Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

    Directory of Open Access Journals (Sweden)

    S. H. Choi

    2015-03-01

    Full Text Available We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC and intramuscular preadipocytes (IPA were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS/Dulbecco’s Modified Eagle Medium (DMEM and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC or 5% FBS/DMEM (IPA with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001 carnitine palmitoyltransferase-1 beta (CPT1β gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases. Oleic and linoleic acid decreased (p = 0.001 stearoyl-CoA desaturase (SCD gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  10. Co-culture with bone marrow stromal cells protects PC12 neuronal cells from tumor necrosis factor-α-induced apoptosis by inhibiting the tumor necrosis factor receptor/caspase signaling pathway.

    Science.gov (United States)

    Li, Li; Wang, Jing; Tang, Ling; Yu, Xin; Sui, Yi; Zhang, Chaodong

    2015-07-01

    Bone marrow stromal cells (BMSCs), derived from the mesoderm, have been applied in the repair and reconstruction of injured tissues. The present study was conducted to explore the effects of BMSCs on cell viability of tumor necrosis factor-α (TNF-α)-stimulated PC12 cells. PC12 cells were co-cultured with BMSCs under TNF-α treatment, with normal PC12 cells as controls. Results from an MTT assay indicated that BMSCs significantly increased cell growth and proliferation of TNF-α-treated PC12 cells (survival rates were 56.71 and 76.86% for the positive control (PC) and co-culture group, respectively). Furthermore, Annexin V/propidium iodide staining and flow cytometric analysis demonstrated that TNF-α increased PC12-cell apoptosis from 3.49 to 40.74% in the negative control and PC group, and the apoptotic rate was significantly reduced upon co-culture with BMSCs to 16.97%. In addition, data from reverse transcription-quantitative polymerase chain reaction and western blot analyses illustrated that TNF-α-induced upregulation in TNF receptor (TNFR)-1 (TNFR1) and caspase-8 expression in PC12 cells were partially reversed by co-culture with BMSCs. In conclusion, the present study suggested that BMSCs protect PC12 cells against stimulation with TNF-α, which is partially mediated through the TNFR/caspase signaling pathway. The results of the present study also suggested a therapeutic use of BMSCs in clinical neurodegenerative diseases.

  11. Mouse preantral follicle growth in 3D co-culture system using human menstrual blood mesenchymal stem cell.

    Science.gov (United States)

    Rajabi, Zahra; Yazdekhasti, Hossein; Noori Mugahi, Seyed Mohammad Hossein; Abbasi, Mehdi; Kazemnejad, Somaieh; Shirazi, Abolfazl; Majidi, Masoumeh; Zarnani, Amir-Hassan

    2018-03-01

    Follicle culture provides a condition which can help investigators to evaluate various aspects of ovarian follicle growth and development and impact of different components and supplementations as well as presumably application of follicle culture approach in fertility preservation procedures. Mesenchymal Stem Cells (MSCs), particularly those isolated from menstrual blood has the potential to be used as a tool for improvement of fertility. In the current study, a 3D co-culture system with mice preantral follicles and human Menstrual Blood Mesenchymal Stem Cells (MenSCs) using either collagen or alginate beads was designed to investigate whether this system allows better preantral follicles growth and development. Results showed that MenSCs increase the indices of follicular growth including survival rate, diameter, and antrum formation as well as the rate of in vitro maturation (IVM) in both collagen and alginates beads. Although statistically not significant, alginate was found to be superior in terms of supporting survival rate and antrum formation. Hormone assay demonstrated that the amount of secreted 17 β-estradiol and progesterone in both 3D systems increased dramatically after 12 days, with the highest levels in system employing MenSCs. Data also demonstrated that relative expression of studied genes increased for Bmp15 and Gdf9 and decreased for Mater when follicles were cultured in the presence of MenSCs. Collectively, results of the present study showed that MenSCs could improve indices of follicular growth and maturation in vitro. Further studies are needed before a clinical application of MenSCs-induced IVM is considered. Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. All rights reserved.

  12. Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches

    Czech Academy of Sciences Publication Activity Database

    Berridge, M.V.; McConnell, M. J.; Grasso, C.; Bajziková, Martina; Kovářová, Jaromíra; Neužil, Jiří

    2016-01-01

    Roč. 38, June (2016), s. 75-82 ISSN 0959-437X R&D Projects: GA ČR(CZ) GAP301/10/1937; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : MESENCHYMAL STEM-CELLS * GENE-TRANSFER * SUPPORTING ASPARTATE BIOSYNTHESIS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.825, year: 2016

  13. [An experimental study of coculture of esophageal mucosa epithelial cells with SIS and their biological characteristics].

    Science.gov (United States)

    Tan, Bo; Wei, Renqian; Yang, Zhiming; Li, Xiuqun; Han, Ping; Zhi, Wei; Xie, Huiqi

    2008-06-01

    To explore an effective method to cultivate esophageal mucosa epithelial cells (EMECs) of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. The primary culture of canine EMECs arranged like slabstone. Immunohistochemical staining of CK-19 of the 2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. EMECs were polygon in shape and arranged like slabstone, and formed a single layer on the surface of SIS. The cells were contacted closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, and showed laminate arrangement. With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS shows good biocompatibility and can be used as a good scaffold material in the tissue engineered esophagus.

  14. A co-culture system with an organotypic lung slice and an immortal alveolar macrophage cell line to quantify silica-induced inflammation.

    Directory of Open Access Journals (Sweden)

    Falk Hofmann

    Full Text Available There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages of wild type and P2rx7-/- mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7-/- mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7-/- mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7-/- mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.

  15. A novel electrospun biphasic scaffold provides optimal three-dimensional topography for in vitro co-culture of airway epithelial and fibroblast cells.

    Science.gov (United States)

    Morris, G E; Bridge, J C; Brace, L A; Knox, A J; Aylott, J W; Brightling, C E; Ghaemmaghami, A M; Rose, F R A J

    2014-09-01

    Conventional airway in vitro models focus upon the function of individual structural cells cultured in a two-dimensional monolayer, with limited three-dimensional (3D) models of the bronchial mucosa. Electrospinning offers an attractive method to produce defined, porous 3D matrices for cell culture. To investigate the effects of fibre diameter on airway epithelial and fibroblast cell growth and functionality, we manipulated the concentration and deposition rate of the non-degradable polymer polyethylene terephthalate to create fibres with diameters ranging from nanometre to micrometre. The nanofibre scaffold closely resembles the basement membrane of the bronchiole mucosal layer, and epithelial cells cultured at the air-liquid interface on this scaffold showed polarized differentiation. The microfibre scaffold mimics the porous sub-mucosal layer of the airway into which lung fibroblast cells showed good penetration. Using these defined electrospinning parameters we created a biphasic scaffold with 3D topography tailored for optimal growth of both cell types. Epithelial and fibroblast cells were co-cultured onto the apical nanofibre phase and the basal microfibre phase respectively, with enhanced epithelial barrier formation observed upon co-culture. This biphasic scaffold provides a novel 3D in vitro platform optimized to mimic the different microenvironments the cells encounter in vivo on which to investigate key airway structural cell interactions in airway diseases such as asthma.

  16. Streptococcus mitis induces conversion of Helicobacter pylori to coccoid cells during co-culture in vitro.

    Science.gov (United States)

    Khosravi, Yalda; Dieye, Yakhya; Loke, Mun Fai; Goh, Khean Lee; Vadivelu, Jamuna

    2014-01-01

    Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptococcus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.

  17. Streptococcus mitis induces conversion of Helicobacter pylori to coccoid cells during co-culture in vitro.

    Directory of Open Access Journals (Sweden)

    Yalda Khosravi

    Full Text Available Helicobacter pylori (H. pylori is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptococcus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.

  18. Exogenous Nkx2.5- or GATA-4-transfected rabbit bone marrow mesenchymal stem cells and myocardial cell co-culture on the treatment of myocardial infarction in rabbits.

    Science.gov (United States)

    Li, Pu; Zhang, Lei

    2015-08-01

    The present study aimed to investigate the effects of Nkx2.5 or GATA-4 transfection with myocardial extracellular environment co-culture on the transformation of bone marrow mesenchymal stem cells (BMSCs) into differentiated cardiomyocytes. Nkx2.5 or GATA-4 were transfected into myocardial extracellular environment co-cultured BMSCs, and then injected into the periphery of infarcted myocardium of a myocardial infarction rabbit model. The effects of these gene transfections and culture on the infarcted myocardium were observed and the results may provide an experimental basis for the efficient myocardial cell differentiation of BMSCs. The present study also suggested that these cells may provide a source and clinical basis for myocardial injury repair via stem cell transplantation. The present study examined whether Nkx2.5 or GATA-4 exogenous gene transfection with myocardial cell extracellular environment co-culture were able to induce the differentiation of BMSCs into cardiac cells. In addition, the effect of these transfected BMSCs on the repair of the myocardium following myocardial infarction was determined using New Zealand rabbit models. The results demonstrated that myocardial cell differentiation was significantly less effective following exogenous gene transfection of Nkx2.5 or GATA-4 alone compared with that of transfection in combination with extracellular environment co-culture. In addition, the results of the present study showed that exogenous gene transfection of Nkx2.5 or GATA-4 into myocardial cell extracellular environment co-cultured BMSCs was able to significantly enhance the ability to repair, mitigating the death of myocardial cells and activation of the myocardium in rabbits with myocardial infarction compared with those of the rabbits transplanted with untreated BMSCs. In conclusion, the exogenous Nkx2.5 and GATA-4 gene transfection into myocardial extracellular environment co-cultured BMSCs induced increased differentiation into myocardial

  19. In vitro co-culture experiments on prostate cancer and small intestine cells irradiated with carbon ions and x-rays

    International Nuclear Information System (INIS)

    Neubeck, C. von; Weyrather, W.-K.; Durante, M.

    2009-01-01

    Intensity modulated radiotherapy (IMRT) delivers the dose in many small irradiation fields of different beam direction to achieve a 3 dimensional tumour conformal dose overlapping with a maximum of normal tissue protection. In 2006 a study was started at GSI to treat prostate cancer patients with a boost irradiation of carbon ions in combination with an IMRT treatment administered at the Uniklinikum Heidelberg. The carbon ions are delivered in two opposing fields. So IMRT irradiation includes more normal tissue than carbon ion treatment but even here parts of the rectum and the bladder are in the irradiated field. This raises the question whether the irradiated tumor cells influence the normal cells (irradiated/ unirradiated) but also whether the normal irradiated cells influences normal tissue in a different way for carbon and photon irradiation. To study this problem, we established an in vitro co-culture model of prostate cancer and small intestine cells of the rat to simulate the patient treatment situation for analyzing tissue reaction exemplary. For characterization of the cells lines the parameters alpha and beta (linear quadratic model) for clonogenic survival were determined for x-rays and for carbon ions of different energies. For co-culture experiments unirradiated and irradiated cells were seeded together and the survival was analyzed

  20. In vitro co-culture models to evaluate acute cytotoxicity of individual and combined mycotoxin exposures on Caco-2, THP-1 and HepaRG human cell lines.

    Science.gov (United States)

    Smith, Marie-Caroline; Gheux, Alexiane; Coton, Monika; Madec, Stéphanie; Hymery, Nolwenn; Coton, Emmanuel

    2018-02-01

    Deoxynivalenol (DON) and zearalenone (ZEA) are mycotoxins primarily produced by Fusarium species and commonly co-occur in European grains. Some in vitro studies reported synergistic combined effects on cell viability reduction for these two natural food contaminants. However, most of these studies were carried out on conventional cell culture systems involving only one cell type and thus did not include cell-cell communication that is closer to in vivo conditions. In this context, we developed easy bi- and tri-culture systems using the Caco-2 (intestinal epithelial cells), THP-1 (monocytes) and HepaRG (hepatic cells) human cell lines in a proliferating state. Individual and combined cytotoxic effects of DON and ZEA were then assessed using co-cultures during 48 h. In bi-culture systems, results showed that only the highest tested dose of ZEA (IC 30 ) induced a significant reduction in THP-1 viability with both Caco-2 and HepaRG cells cultured in transwells above. On the contrary, only the highest tested dose of DON (IC 30 ) significantly affected HepaRG cell viability located under the Caco-2 cell monolayer. In addition, the DON + ZEA combination seemed to induce higher cytotoxicity than each toxin alone. Mycotoxin quantification in the abluminal compartment by Q-TOF LC-MS suggested uptake of both mycotoxins by the different cell lines. According to the co-culturing cell type, possible cell-cell interactions were also observed. Finally, in the tri-culture system, no cytotoxic effects were observed, regardless of the treatment. These findings highlighted the importance of the proposed models to better decipher toxicological impacts of mycotoxins on more complex cellular systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Co-culture of neural crest stem cells (NCSC and insulin producing beta-TC6 cells results in cadherin junctions and protection against cytokine-induced beta-cell death.

    Directory of Open Access Journals (Sweden)

    Anongnad Ngamjariyawat

    Full Text Available PURPOSE: Transplantation of pancreatic islets to Type 1 diabetes patients is hampered by inflammatory reactions at the transplantation site leading to dysfunction and death of insulin producing beta-cells. Recently we have shown that co-transplantation of neural crest stem cells (NCSCs together with the islet cells improves transplantation outcome. The aim of the present investigation was to describe in vitro interactions between NCSCs and insulin producing beta-TC6 cells that may mediate protection against cytokine-induced beta-cell death. PROCEDURES: Beta-TC6 and NCSC cells were cultured either alone or together, and either with or without cell culture inserts. The cultures were then exposed to the pro-inflammatory cytokines IL-1β and IFN-γ for 48 hours followed by analysis of cell death rates (flow cytometry, nitrite production (Griess reagent, protein localization (immunofluorescence and protein phosphorylation (flow cytometry. RESULTS: We observed that beta-TC6 cells co-cultured with NCSCs were protected against cytokine-induced cell death, but not when separated by cell culture inserts. This occurred in parallel with (i augmented production of nitrite from beta-TC6 cells, indicating that increased cell survival allows a sustained production of nitric oxide; (ii NCSC-derived laminin production; (iii decreased phospho-FAK staining in beta-TC6 cell focal adhesions, and (iv decreased beta-TC6 cell phosphorylation of ERK(T202/Y204, FAK(Y397 and FAK(Y576. Furthermore, co-culture also resulted in cadherin and beta-catenin accumulations at the NCSC/beta-TC6 cell junctions. Finally, the gap junction inhibitor carbenoxolone did not affect cytokine-induced beta-cell death during co-culture with NCSCs. CONCLUSION: In summary, direct contacts, but not soluble factors, promote improved beta-TC6 viability when co-cultured with NCSCs. We hypothesize that cadherin junctions between NCSC and beta-TC6 cells promote powerful signals that maintain beta-cell

  2. Proteomic View of the Crosstalk between Lactobacillus mucosae and Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics

    Directory of Open Access Journals (Sweden)

    Edward Alain B. Pajarillo

    2017-12-01

    Full Text Available Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2 after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host–microbe crosstalk and response. These findings will provide novel insights into host–bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

  3. Proteomic View of the Crosstalk betweenLactobacillus mucosaeand Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics.

    Science.gov (United States)

    Pajarillo, Edward Alain B; Kim, Sang Hoon; Valeriano, Valerie Diane; Lee, Ji Yoon; Kang, Dae-Kyung

    2017-01-01

    Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2) after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways) were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host-microbe crosstalk and response. These findings will provide novel insights into host-bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

  4. Use of monocyte/endothelial cell co-cultures (in vitro) and a subcutaneous implant mouse model (in vivo) to evaluate a degradable polar hydrophobic ionic polyurethane.

    Science.gov (United States)

    McDonald, Sarah M; Matheson, Loren A; McBane, Joanne E; Kuraitis, Drew; Suuronen, Erik; Santerre, Joseph Paul; Labow, Rosalind S

    2011-12-01

    Potential benefits of co-culturing monocytes (MC) with vascular smooth muscle cells have been reported on for tissue engineering applications with a degradable, polar, hydrophobic, and ionic polyurethane (D-PHI). Since the interaction of MC and endothelial cells (EC) within the blood vessel endothelium is also a process of wound repair it was of interest to investigate their function when cultured on the synthetic D-PHI materials, prior to considering the materials' use in vascular engineering. The co-culture (MC/EC) in vitro studies were carried out on films in 96 well plates and porous scaffold disks were prepared for implant studies in an in vivo subcutaneous mouse model. After 7 days in culture, the MC/EC condition was equal to EC growth but had lower esterase activity (a measure of degradative potential), no pro-inflammatory TNF-α and a relatively high anti-inflammatory IL-10 release while the ECs maintained their functional marker CD31. After explantation of the porous scaffolds, a live/dead stain showed that the cells infiltrating the scaffolds were viable and histological stains (May-Grunwald, Trichrome) demonstrated tissue in growth and extracellular matrix synthesis. Lysates from the implant scaffolds analyzed with a cytokine antibody array showed decreased pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF), increased anti-inflammatory cytokines (IL-10, IL-13, TNF-RI), and increased chemotactic cytokines (MCP-1, MCP-5, RANTES). The low foreign body response elicited by D-PHI when implanted in vivo supported the in vitro studies (EC and MC co-culture), demonstrating that D-PHI promoted EC growth along with an anti-inflammatory MC, further demonstrating its potential as a tissue engineering scaffold for vascular applications. Copyright © 2011 Wiley Periodicals, Inc.

  5. [Effects of Xiaoyao Powder Extract in co-culture model of melanoma cells and keratinocytes on expression of tyrosinase and its related protein].

    Science.gov (United States)

    Zhang, Ning; Liu, Hui; Zhang, Ming-lei; Liu, Bin; Ren, Yan-dong; Wang, Xue; Li, Jian-min

    2014-06-01

    To observe the effects and mechanism of Xiaoyao Powder extract on the content of melanin in co-culture model of melanoma cells and keratinocytes. Eluting components of Xiaoyao Powder was collected by AB-8 macroporous resin column. Different concentration extracts of Xiaoyao Powder were added into the co-culture model of A375 melanoma cells and HaCat ke- ratinocytes. Hunt method was used to detected the content of melanin. RT-PCR assay was used to detect the effects of the extract of Xi- aoyao Powder on the TYR,TRP-1 and TRP-2 mRNA expression in A375 melanoma cells. Compared with the control group,the extract of Xiaoyao Powder down-regulated content of melanin and mRNA expression of TYR,TRP-1 and TRP-2 in A375 melanoma cells by 82.23% ,93. 01% and 29. 11% ,23.78% ,20. 05% ;25. 13% ,15.02% ,11.64% ,respectively(P Powder can decrease the melanogenesis by down-regulating the mRNA expression of tyrosinase and its related protein.

  6. Bioaccessibility, bioavailability, and anti-inflammatory effects of anthocyanins from purple root vegetables using mono- and co-culture cell models.

    Science.gov (United States)

    Zhang, Hua; Hassan, Yousef I; Renaud, Justin; Liu, Ronghua; Yang, Cheng; Sun, Yong; Tsao, Rong

    2017-10-01

    Immune-inflammatory signaling and metabolic effects are the main pillars for bioactivity of anthocyanins derived from highly pigmented root vegetables. This study aims to assess the bioaccessibility and bioavailability of purple carrot and potato derived anthocyanins and the molecular mechanisms of their ability to ameliorate cellular inflammation in a mono- and co-culture cell models. An in vitro gastrointestinal model was used and demonstrated bioaccessibility of 44.62 and 71.8% for anthocyanins of purple carrot and potato, respectively. These accessible anthocyanins significantly inhibited cellular inflammation in Caco-2 cells. Intact cyanidinglycoside or petunidinglycoside (respectively from carrots and potatoes) were transported across a transmembrane cell model and detected by LC-MS/MS. Computational docking and glucose uptake analyses suggested uptake of anthocyanins was mediated by hexose transporters. Subsequent experiment using an inflamed Caco-2 BBe1/THP-1 co-culture cell model showed these transported anthocyanins inhibited IL-8 and TNF-α secretion,and expression of pro-inflammatory cytokines by blocking NF-κB, and MAPK mediated inflammatory cellular signaling cascades, but with varying degrees due to structural features. Anthocyanins from purple carrots and potatoes possess a promising anti-inflammatory effect in model gut system. They can be absorbed and act differently but are in general beneficial for inflammation-mediated diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Inverse-power-law behavior of cellular motility reveals stromal-epithelial cell interactions in 3D co-culture by OCT fluctuation spectroscopy.

    Science.gov (United States)

    Oldenburg, Amy L; Yu, Xiao; Gilliss, Thomas; Alabi, Oluwafemi; Taylor, Russell M; Troester, Melissa A

    2015-10-20

    The progression of breast cancer is known to be affected by stromal cells within the local microenvironment. Here we study the effect of stromal fibroblasts on the in-place motions (motility) of mammary epithelial cells within organoids in 3D co-culture, inferred from the speckle fluctuation spectrum using optical coherence tomography (OCT). In contrast to Brownian motion, mammary cell motions exhibit an inverse power-law fluctuation spectrum. We introduce two complementary metrics for quantifying fluctuation spectra: the power-law exponent and a novel definition of the motility amplitude, both of which are signal- and position-independent. We find that the power-law exponent and motility amplitude are positively ( p <0.001) and negatively ( p <0.01) correlated with the density of stromal cells in 3D co-culture, respectively. We also show how the hyperspectral data can be visualized using these metrics to observe heterogeneity within organoids. This constitutes a simple and powerful tool for detecting and imaging cellular functional changes with OCT.

  8. Inverse-power-law behavior of cellular motility reveals stromal–epithelial cell interactions in 3D co-culture by OCT fluctuation spectroscopy

    Science.gov (United States)

    Oldenburg, Amy L.; Yu, Xiao; Gilliss, Thomas; Alabi, Oluwafemi; Taylor, Russell M.; Troester, Melissa A.

    2015-01-01

    The progression of breast cancer is known to be affected by stromal cells within the local microenvironment. Here we study the effect of stromal fibroblasts on the in-place motions (motility) of mammary epithelial cells within organoids in 3D co-culture, inferred from the speckle fluctuation spectrum using optical coherence tomography (OCT). In contrast to Brownian motion, mammary cell motions exhibit an inverse power-law fluctuation spectrum. We introduce two complementary metrics for quantifying fluctuation spectra: the power-law exponent and a novel definition of the motility amplitude, both of which are signal- and position-independent. We find that the power-law exponent and motility amplitude are positively (p<0.001) and negatively (p<0.01) correlated with the density of stromal cells in 3D co-culture, respectively. We also show how the hyperspectral data can be visualized using these metrics to observe heterogeneity within organoids. This constitutes a simple and powerful tool for detecting and imaging cellular functional changes with OCT. PMID:26973862

  9. In vitro generation of whole osteochondral constructs using rabbit bone marrow stromal cells, employing a two-chambered co-culture well design.

    Science.gov (United States)

    Chen, Kelei; Ng, Kian Siang; Ravi, Sujata; Goh, James C H; Toh, Siew Lok

    2016-04-01

    The regeneration of whole osteochondral constructs with a physiological structure has been a significant issue, both clinically and academically. In this study, we present a method using rabbit bone marrow stromal cells (BMSCs) cultured on a silk-RADA peptide scaffold in a specially designed two-chambered co-culture well for the generation of multilayered osteochondral constructs in vitro. This specially designed two-chambered well can simultaneously provide osteogenic and chondrogenic stimulation to cells located in different regions of the scaffold. We demonstrated that this co-culture approach could successfully provide specific chemical stimulation to BMSCs located on different layers within a single scaffold, resulting in the formation of multilayered osteochondral constructs containing cartilage-like and subchondral bone-like tissue, as well as the intermediate osteochondral interface. The cells in the intermediate region were found to be hypertrophic chondrocytes, embedded in a calcified extracellular matrix containing glycosaminoglycans and collagen types I, II and X. In conclusion, this study provides a single-step approach that highlights the feasibility of rabbit BMSCs as a single-cell source for multilayered osteochondral construct generation in vitro. Copyright © 2013 John Wiley & Sons, Ltd.

  10. Viable and morphologically normal boar spermatozoa alter the expression of heat-shock protein genes in oviductal epithelial cells during co-culture in vitro.

    Science.gov (United States)

    Yeste, Marc; Holt, William V; Bonet, Sergi; Rodríguez-Gil, Joan E; Lloyd, Rhiannon E

    2014-09-01

    The principal aim of this study was to determine if boar spermatozoa influence the expression of four selected chaperone and heat-shock protein (HSP) genes-namely clusterin (CLU), HSP90AA1, HSPA5, and HSPA8-in oviductal epithelial cells (OECs) during in vitro co-culture. All corresponding proteins of these genes were previously identified in a sperm-interacting, 70-kDa soluble fraction derived from apical plasma membranes of OECs. The present study also sought to determine whether or not: (i) spermatozoa must directly bind to OEC for an effect on gene expression to be elicited and (ii) reproductive and nonreproductive epithelial cell types (LLC-PK1, pig kidney) respond equivalently, in terms of alterations in chaperone and HSP gene expression, during co-culture with sperm. Spermatozoa induced a significant upregulation (P culture when they were in direct contact with epithelial cells. Conversely, no upregulation of HSP transcription was observed when spermatozoa did not directly bind to OECs. Spermatozoa also induced a significant upregulation (P caused by direct contact between spermatozoa and OECs, rather than nonreproductive epithelial cells, suggests HSPs could play an integral role in the modulation of sperm function in the oviductal reservoir. © 2014 Wiley Periodicals, Inc.

  11. Semiautomatic High-Content Analysis of Complex Images from Cocultures of Vascular Smooth Muscle Cells and Macrophages: A CellProfiler Showcase.

    Science.gov (United States)

    Roeper, Matthias; Braun-Dullaeus, Ruediger C; Weinert, Sönke

    2017-08-01

    Automatization in microscopy, cell culture, and the ease of digital imagery allow obtainment of more information from single samples and upscaling of image-based analysis to high-content approaches. Simple segmentation algorithms of biological imagery are nowadays widely spread in biomedical research, but processing of complex sample structures, for example, variable sample compositions, cell shapes, and sizes, and rare events remains a difficult task. As there is no perfect method for image segmentation and fully automatic image analysis of complex content, we aimed to succeed by identification of unique and reliable features within the sample. Through exemplary use of a coculture of vascular smooth muscle cells (VSMCs) and macrophages (MPs), we demonstrate how rare interactions within this highly variable sample type can be analyzed. Because of limitations in immunocytochemistry in our specific setup, we developed a semiautomatic approach to examine the interaction of lipid-laden MPs with VSMCs under hypoxic conditions based on nuclei morphology by high-content analysis using the open-source software CellProfiler ( www.cellprofiler.org ). We provide evidence that, in comparison with fully automatic analysis, a low threshold within the analysis workflow and subsequent manual control save time, while providing more objective and reliable results.

  12. Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells

    Science.gov (United States)

    Castiaux, Virginie; Laloux, Laurie; Schneider, Yves-Jacques; Mahillon, Jacques

    2016-01-01

    B. cereus is an opportunistic foodborne pathogen able to cause diarrhoea. However, the diarrhoeal potential of a B. cereus strain remains difficult to predict, because no simple correlation has yet been identified between the symptoms and a unique or a specific combination of virulence factors. In this study, 70 B. cereus strains with different origins (food poisonings, foods and environment) have been selected to assess their enterotoxicity. The B. cereus cell-free supernatants have been tested for their toxicity in vitro, on differentiated (21 day-old) Caco-2 cells, using their ATP content, LDH release and NR accumulation. The genetic determinants of the main potential enterotoxins and virulence factors (ces, cytK, entFM, entS, hbl, nhe, nprA, piplC and sph) have also been screened by PCR. This analysis showed that none of these genes was able to fully explain the enterotoxicity of B. cereus strains. Additionally, in order to assess a possible effect of the mucus layer in vitro, a cytotoxicity comparison between a monoculture (Caco-2 cells) and a co-culture (Caco-2 and HT29-MTX mucus-secreting cells) model has been performed with selected B. cereus supernatants. It appeared that, in these conditions, the mucus layer had no notable influence on the cytotoxicity of B. cereus supernatants. PMID:27827957

  13. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

    Directory of Open Access Journals (Sweden)

    Schneider Yves-Jacques

    2006-05-01

    Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

  14. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

    Science.gov (United States)

    Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

    2006-01-01

    Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

  15. From the Cover: AstrocytesAre Protective Against Chlorpyrifos Developmental Neurotoxicity in Human Pluripotent Stem Cell-Derived Astrocyte-Neuron Cocultures.

    Science.gov (United States)

    Wu, Xian; Yang, Xiangkun; Majumder, Anirban; Swetenburg, Raymond; Goodfellow, Forrest T; Bartlett, Michael G; Stice, Steven L

    2017-06-01

    Human neural progenitor cells are capable of independent, directed differentiation into astrocytes, oligodendrocytes and neurons and thus offer a potential cell source for developmental neurotoxicity (DNT) systems. Human neural progenitor-derived astrocyte-neuron cocultured at defined ratios mimic cellular heterogeneity and interaction in the central nervous system. Cytochrome P450 enzymes are expressed at a relatively high level in astrocytes and may play a critical role in the biotransformation of endogenous or exogenous compounds, including chlorpyrifos, an organophosphate insecticide that affects the central nervous system. P450 enzymes metabolize chlorpyrifos to chlorpyrifos-oxon, which is then metabolized primarily to 3, 5, 6-trichloropyridinol in addition to diethylphosphate and diethylthiophosphate. These end metabolites are less neurotoxic than chlorpyrifos and chlorpyrifos-oxon. Our objective was to identify the interactive role of astrocytes and neurons in chlorpyrifos-induced human DNT. In neuron-only cultures, chlorpyrifos inhibited neurite length, neurite number and branch points per neuron in a dose-dependent manner during a 48 h exposure, starting at 10 μM. However, in astrocyte-neuron cocultures, astrocytes protected neurons from the effects of chlorpyrifos at higher concentrations, up to and including 30 μM chlorpyrifos and endogenous astrocyte P450 enzymes effectively metabolized chlorpyrifos. The P450 inhibitor SKF525A partly negated the protective effect of astrocytes, allowing reduction in branch points with chlorpyrifos (10 μM). Thus, the scalable and defined astrocyte-neuron cocultures model that we established here has potentially identified a role for P450 enzymes in astrocytic neuroprotection against chlorpyrifos and provides a novel model for addressing DNT in a more accurate multicellular environment. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For

  16. Co-culture with podoplanin+ cells protects leukemic blast cells with leukemia-associated antigens in the tumor microenvironment.

    Science.gov (United States)

    Lee, Ji Yoon; Han, A-Reum; Lee, Sung-Eun; Min, Woo-Sung; Kim, Hee-Je

    2016-05-01

    Podoplanin+ cells are indispensable in the tumor microenvironment. Increasing evidence suggests that podoplanin may support the growth and metastasis of solid tumors; however, to the best of our knowledge no studies have determined whether or not podoplanin serves a supportive role in acute myeloid leukemia (AML). The effects of co‑culture with podoplanin+ cells on the cellular activities of the leukemic cells, such as apoptosis and cell proliferation, in addition to the expression of podoplanin in leukemic cells, were investigated. Due to the fact that genetic abnormalities are the primary cause of leukemogenesis, the overexpression of the fibromyalgia‑like tyrosine kinase‑3 gene in colony forming units was also examined following cell sorting. Podoplanin+ cells were found to play a protective role against apoptosis in leukemic cells and to promote cell proliferation. Tumor‑associated antigens, including Wilms' tumor gene 1 and survivin, were increased when leukemic cells were co‑cultured with podoplanin+ cells. In combination, the present results also suggest that podoplanin+ cells can function as stromal cells for blast cell retention in the AML tumor microenvironment.

  17. Co-Culture with Human Osteoblasts and Exposure to Extremely Low Frequency Pulsed Electromagnetic Fields Improve Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sabrina Ehnert

    2018-03-01

    Full Text Available Human adipose-derived mesenchymal stem cells (Ad-MSCs have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs. Thus, the project aimed at (i investigating whether co-culture conditions of human osteoblasts (OBs and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz; (iii and the effect of these ELF-PEMFs on human osteoclasts (OCs. Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz could enhance bone formation, while the higher frequency (26 Hz could enhance bone remodeling.

  18. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF- beta1 and/or CPT-11.

    Science.gov (United States)

    Paduch, Roman; Jakubowicz-Gil, Joanna; Kandefer-Szerszen, Martyna

    2009-12-01

    We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the influence of recombinant human transforming growth factor beta1 (rhTGF-beta1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO). An immunoblotting analysis with densitometry showed that rhTGF-beta1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofibroblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable. Direct interaction of colon tumour spheroids with normal myofibroblasts caused a significant, tumour-grade dependent increase in IL-6 production. Production of IL-6 was significantly lowered by rhTGF-beta1 and/or CPT-11. Tumour cell spheroids cultivated alone produced larger amounts of NO than normal cells. In co-culture, the level of the radical decreased compared with the sum of NO produced by the monocultures of the two types of cells. rhTGF-beta1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures. In conclusion, direct interactions between tumour and normal cells influence the expression of HSP27, HSP72 and MRP, and alter IL-6 and NO production. rhTGF-beta1 and/or CPT-11 may potentate resistance to chemotherapy by increasing HSP and MRP expression but, on the other hand, they may limit tumour cell spread by decreasing the level of some soluble mediators of inflammation (IL-6 and NO).

  19. Depsidones inhibit aromatase activity and tumor cell proliferation in a co-culture of human primary breast adipose fibroblasts and T47D breast tumor cells.

    Science.gov (United States)

    Chottanapund, Suthat; Van Duursen, M B M; Zwartsen, Anne; Timtavorn, Supatchaya; Navasumrit, Panida; Kittakoop, Prasat; Sureram, Sanya; Ruchirawat, Mathuros; Van den Berg, Martin

    2017-01-01

    Naturally occurring depsidones from the marine fungus Aspergillus unguis are known to have substantial anti-cancer activity, but their mechanism of action remains elusive. The purpose of this study was to examine the anti-aromatase activity of two common depsidones, unguinol and aspergillusidone A, in a co-culture system of human primary breast adipose fibroblasts and hormonal responsive T47D breast tumor cells. Using this in vitro model it was shown that these depsidones inhibit the growth of T47D tumor cells most likely via inhibition of aromatase (CYP19) activity. The IC 50 values of these depisidones were compared with the aromatase inhibitors letrozole and exemestane. Letrozole and exemestane had IC 50 values of respectively, 0.19 and 0.14 μM, while those for Unguinol and Aspergillusidone A were respectively, 9.7 and 7.3 μM. Our results indicate that among the depsidones there maybe aromatase inhibitors with possible pharmacotherapeutical relevance.

  20. Glia-Neuron Interactions in the Retina Can Be Studied in Cocultures of Muller Cells and Retinal Ganglion Cells

    DEFF Research Database (Denmark)

    Skytt, D. M.; Toft-Kehler, A. K.; Braendstrup, C. T.

    2016-01-01

    Glia-neuron partnership is important for inner retinal homeostasis and any disturbances may result in retinal ganglion cell (RGC) death. Müller cells support RGCs with essential functions such as removing excess glutamate and providing energy sources. The aim was to explore the impact of Müller c...

  1. Vaccine-Mediated Mechanisms Controlling Replication of Francisella tularensis in Human Peripheral Blood Mononuclear Cells Using a Co-culture System

    Directory of Open Access Journals (Sweden)

    Kjell Eneslätt

    2018-02-01

    Full Text Available Cell-mediated immunity (CMI is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to

  2. Fermentative and growth performances of Dekkera bruxellensis in different batch systems and the effect of initial low cell counts in co-cultures with Saccharomyces cerevisiae.

    Science.gov (United States)

    Meneghin, Maria Cristina; Bassi, Ana Paula Guarnieri; Codato, Carolina Brito; Reis, Vanda Renata; Ceccato-Antonini, Sandra Regina

    2013-08-01

    Dekkera bruxellensis is a multifaceted yeast present in the fermentative processes used for alcoholic beverage and fuel alcohol production - in the latter, normally regarded as a contaminant. We evaluated the fermentation and growth performance of a strain isolated from water in an alcohol-producing unit, in batch systems with/without cell recycling in pure and co-cultures with Saccharomyces cerevisiae. The ethanol resistance and aeration dependence for ethanol/acid production were verified. Ethanol had an effect on the growth of D. bruxellensis in that it lowered or inhibited growth depending on the concentration. Acid production was verified in agitated cultures either with glucose or sucrose, but more ethanol was produced with glucose in agitated cultures. Regardless of the batch system, low sugar consumption and alcohol production and expressive growth were found with D. bruxellensis. Despite a similar ethanol yield compared to S. cerevisiae in the batch system without cell recycling, ethanol productivity was approximately four times lower. However, with cell recycling, ethanol yield was almost half that of S. cerevisiae. At initial low cell counts of D. bruxellensis (10 and 1000 cells/ml) in co-cultures with S. cerevisiae, a decrease in fermentative efficiency and a substantial growth throughout the fermentative cycles were displayed by D. bruxellensis. Due to the peculiarity of cell repitching in Brazilian fermentation processes, D. bruxellensis is able to establish itself in the process, even when present in low numbers initially, substantially impairing bioethanol production due to the low ethanol productivity, in spite of comparable ethanol yields. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System

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    Izuru Mizoguchi

    2017-08-01

    Full Text Available The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel in vitro assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs derived from human peripheral blood CD14+ monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the in vivo situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L, interleukin (IL-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD

  4. Prediction of Chemical Respiratory and Contact Sensitizers by OX40L Expression in Dendritic Cells Using a Novel 3D Coculture System.

    Science.gov (United States)

    Mizoguchi, Izuru; Ohashi, Mio; Chiba, Yukino; Hasegawa, Hideaki; Xu, Mingli; Owaki, Toshiyuki; Yoshimoto, Takayuki

    2017-01-01

    The use of animal models in chemical safety testing will be significantly limited due to the recent introduction of the 3Rs principle of animal experimentation in research. Although several in vitro assays to predict the sensitizing potential of chemicals have been developed, these methods cannot distinguish chemical respiratory sensitizers and skin sensitizers. In the present study, we describe a novel in vitro assay that can discriminate respiratory sensitizers from chemical skin sensitizers by taking advantage of the fundamental difference between their modes of action, namely the development of the T helper 2 immune response, which is critically important for respiratory sensitization. First, we established a novel three-dimensional (3D) coculture system of human upper airway epithelium using a commercially available scaffold. It consists of human airway epithelial cell line BEAS-2B, immature dendritic cells (DCs) derived from human peripheral blood CD14 + monocytes, and human lung fibroblast cell line MRC-5. Respective cells were first cultured in individual scaffolds and subsequently assembled into a 3D multi-cell tissue model to more closely mimic the in vivo situation. Then, three typical chemicals that are known respiratory sensitizers (ortho-phthaldialdehyde, hexamethylene diisocyanate, and trimellitic anhydride) and skin sensitizers (oxazolone, formaldehyde, and dinitrochlorobenzene) were added individually to the 3D coculture system. Immunohistochemical analysis revealed that DCs do not migrate into other scaffolds under the experimental conditions. Therefore, the 3D structure was disassembled and real-time reverse transcriptase-PCR analysis was performed in individual scaffolds to analyze the expression levels of molecules critical for Th2 differentiation such as OX40 ligand (OX40L), interleukin (IL)-4, IL-10, IL-33, and thymic stromal lymphopoietin. Both sensitizers showed similarly augmented expression of DC maturation markers (e.g., CD86), but among

  5. Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering.

    Science.gov (United States)

    Kremer, Antje; Ribitsch, Iris; Reboredo, Jenny; Dürr, Julia; Egerbacher, Monika; Jenner, Florien; Walles, Heike

    2017-05-01

    Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

  6. Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device.

    Science.gov (United States)

    van der Meer, Andries D; Orlova, Valeria V; ten Dijke, Peter; van den Berg, Albert; Mummery, Christine L

    2013-09-21

    Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here, we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells, human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h, the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell-cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels, inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity, highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary, we have

  7. Advanced cell culture technology for generation of in vivo-like tissue models

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-06-01

    Full Text Available Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011. The scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. This technology has been applied to generate numerous unique types of co-culture model. For example: 1 a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (Fig.1 (Hill et al., 2015; 2 a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (Clarke et al., 2016; 3 formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ECM proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa. These organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. Creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. The addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. Methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in

  8. [Differentiation of directly co-cultured bone marrow mesenchymal stem cells and ligament fibroblasts into ligament cells after induced by transforming growth factor β1 and basic fibroblast growth factor].

    Science.gov (United States)

    Liu, Yi; Zhang, Chenghao; Fan, Qinghong; Sun, Pengpeng; Wu, Shuhong

    2014-11-01

    To investigate the effect of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, proliferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. BMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and proliferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type I and type III were determined by ELISA; and mRNA expressions of collagen types I and III, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. A single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion (> 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P 0.05). The ratios of collagen type I to type III were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type I and type III and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). Directly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation

  9. Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes.

    Science.gov (United States)

    Jang, Jae-Woo; Song, Yeonhwa; Kim, Kang Mo; Kim, Jin-Sun; Choi, Eun Kyung; Kim, Joon; Seo, Haengran

    2016-10-18

    Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in HCC patients. Here, we developed a fluorescence image-based phenotypic screening system in vitro to identify HCC-specific drugs in co-cultures of HCC cells with hepatocytes. To this end, we identified two distinctive markers of HCC, CHALV1 and AFP, which are highly expressed in HCC cell lines and liver cancer patient-derived materials. We applied these markers to an HCC-specific drug screening system. Through pilot screening, we identified three anti-folate compounds that had HCC-specific cytotoxicity. Among them, pyrimethamine exhibited the greatest HCC-specific cytotoxicity. Interestingly, pyrimethamine significantly increased the size and number of lysosomes and subsequently induced the release of cathepsin B from the lysosome to the cytosol, which triggered caspase-3-dependent apoptosis in Huh7 (HCC) but not Fa2N-4 cells (immortalized hepatocytes). Importantly, Fa2N-4 cells had strong resistance to pyrimethamine relative to Huh7 cells in 2D and 3D culture systems. These results demonstrate that this in vitro image-based phenotypic screening platform has the potential to be widely adopted in drug discovery research, since we promptly estimated anticancer activity and hepatotoxicity and elucidated functional roles of pyrimethamine during the apoptosis process in HCC.

  10. Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture.

    Science.gov (United States)

    Kaiser, Andreas; Kale, Ajay; Novozhilova, Ekaterina; Siratirakun, Piyaporn; Aquino, Jorge B; Thonabulsombat, Charoensri; Ernfors, Patrik; Olivius, Petri

    2014-05-30

    Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Co-cultures of human coronary smooth muscle cells and dimethyl sulfoxide-differentiated HL60 cells upregulate ProMMP9 activity and promote mobility-modulation by reactive oxygen species.

    Science.gov (United States)

    Bernard, Yohann; Melchior, Chantal; Tschirhart, Eric; Bueb, Jean-Luc

    2008-10-01

    Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We investigated by co-culture experiments the effects of human coronary smooth muscle cells (HCSMC) on MMPs characteristics and mobility of neutrophil-like dimethyl sulfoxide-differentiated HL60 cells (not equal HL60). The effects of superoxide dismutase (SOD) and catalase were also analyzed. All the studied MMP2 characteristics remained unchanged. HCSMC stimulated MMP9 protein level, activity and mobility of not equal HL60 cells and expressed and secreted a variety of cytokines implicated in atherosclerosis. SOD and catalase increased MMP9 expression, protein level and activity of not equal HL60, but migration of not equal HL60 cells was only decreased by catalase, demonstrating that ROS are more efficient in modulating MMP9 activity of not equal HL60 than their mobility. Finally, HCSMC being able to stimulate not equal HL60, their co-cultures may represent an in vitro approach to study cellular interactions occurring in vivo during atherosclerosis.

  12. Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.

    Science.gov (United States)

    Yasuda, Michiko; Furuyashiki, Takashi; Nakamura, Toshiyuki; Kakutani, Ryo; Takata, Hiroki; Ashida, Hitoshi

    2013-09-01

    Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.

  13. Sunitinib indirectly enhanced anti-tumor cytotoxicity of cytokine-induced killer cells and CD3⁺CD56⁺ subset through the co-culturing dendritic cells.

    Directory of Open Access Journals (Sweden)

    Adisak Wongkajornsilp

    Full Text Available Cytokine-induced killer (CIK cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs. We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6 in DCs at the expense of Th2 inducing phenotype (IL-13 and regulatory phenotype (PD-L1, IDO. Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet and the downregulation of the Th2 signature (GATA-3 and the Th17 marker (RORC on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.

  14. Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co-culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs, human induced pluripotent stem cell-derived MSCs and human embryonic stem cell-derived MSCs.

    Science.gov (United States)

    Chen, Wenchuan; Liu, Xian; Chen, Qianmin; Bao, Chongyun; Zhao, Liang; Zhu, Zhimin; Xu, Hockin H K

    2017-01-18

    Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co-culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co-cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC-MSCs) and embryonic stem cells (hESC-MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary-like structures were successfully formed on CPC in vitro in all four co-cultured groups. Microcapillary lengths increased with time (p cells increased with time (p  0.1). Therefore, hUCMSCs, hiPSC-MSCs and hESC-MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co-cultures of hUVECs with hUCMSCs, hiPSC-MSCs, hESC-MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co-culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  15. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

    Science.gov (United States)

    Helms, Hans Christian; Madelung, Rasmus; Waagepetersen, Helle Sønderby; Nielsen, Carsten Uhd; Brodin, Birger

    2012-05-01

    The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. Copyright © 2012 Wiley Periodicals, Inc.

  16. Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

    International Nuclear Information System (INIS)

    Vickers, Alison E.M.; Sinclair, John R.; Fisher, Robyn L.; Morris, Stephen R.; Way, William

    2010-01-01

    A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (≥ 1000 μM at 48 h) and human tissues (≥ 1000 μM at 48 h, ≥ 750 μM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

  17. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  18. Generation of Hepatocyte-like Cells from Human Induced Pluripotent Stem (iPS) Cells By Co-culturing Embryoid Body Cells with Liver Non-parenchymal Cell Line TWNT-1

    International Nuclear Information System (INIS)

    Javed, M. S.; Yaqoob, N.; Iwamuro, M.; Kobayashi, N.; Fujiwara, T.

    2014-01-01

    Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds. (author)

  19. Characterisation of an in vitro blood-brain barrier model based on primary porcine capillary endothelial cells in monoculture or co-culture with primary rat or porcine astrocytes and pericytes

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Larsen, Annette Burkhart; Moos, Torben

    to in vivo such as efflux transporters, tight junction proteins, and high transendothelial electric resistance (TEER). Primary BCECs are isolated from a variety of mammals such as rats, mice, cattle and pigs. Often bovine and porcine BCECs are cultured in monoculture or in co-culture with rat astrocytes......In vitro blood-brain barrier (BBB) models based on primary brain capillary endothelial cells (BCECs) in monoculture or in co-culture with primary astrocytes and pericytes are often applied for studying physiology of the BBB. Primary BCECs retain many morphological and biochemical properties similar...... obtained from neonatal rats which have been shown to strengthen the barrier properties of the BCECs. In this study, brain endothelial cells (PBECs), astrocytes and pericytes are isolated from pig brains donated by the local abattoir. The brains are from 6 month old domestic pigs. The availability and high...

  20. A Fateful Meeting of Two Phytoplankton Species-Chemical vs. Cell-Cell-Interactions in Co-Cultures of the Green Algae Oocystis marsonii and the Cyanobacterium Microcystis aeruginosa.

    Science.gov (United States)

    Dunker, Susanne; Althammer, Julia; Pohnert, Georg; Wilhelm, Christian

    2017-07-01

    Massive growth of single species of cyanobacteria is a common phenomenon in many eutrophicated waters worldwide. Allelopathic growth control of phytoplankton species is one suggested mechanism, but still controversially discussed. The fact that the synthesis of biological active compounds requires high energy costs and carbon investment for a single cell in contrast to high dilution rates in natural systems questions the universal validity of allelopathic mechanisms, even more as high concentrations of allelopathic substances are often needed in several experiments to cause biological effects. In this study, it was tested, if growth inhibition is induced by chemical signaling alone or via direct cell-cell interaction. As a test system, we used a co-culture of the green algae Oocystis marsonii (Trebouxiophyceae) with the cyanobacterium Microcystis aeruginosa which is known to strongly reduce the growth of the green algal competitor. In this study, direct co-culturing as well as membrane-separated growth chambers were used to test for chemical and contact-mediated interactions. In the membrane-separated chambers, both species can be co-cultivated and a membrane allows the exchange of metabolites. Growth of O. marsonii was only affected in the direct co-cultivation situation, where direct cell-to-cell contact was possible. During direct co-cultivation, deviating cellular traits, namely cell cycle pattern and large cell-aggregate formation of both species, could be detected. These data strongly support the hypothesis of a direct cell-cell-contact necessary for allelopathic growth control in this model system. Such direct contact would allow targeting allelopathic metabolites directly towards the competitor and thereby minimizing dilution effects.

  1. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  2. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  3. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Directory of Open Access Journals (Sweden)

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  4. Anaerobic co-culture of mesenchymal stem cells and anaerobic pathogens - a new in vitro model system.

    Directory of Open Access Journals (Sweden)

    Katja Kriebel

    Full Text Available BACKGROUND: Human mesenchymal stem cells (hMSCs are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens. METHODOLOGY/PRINCIPAL FINDINGS: We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively. In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments. CONCLUSIONS/SIGNIFICANCE: HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of

  5. Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

    Science.gov (United States)

    Mills, Dominic C; Gundogdu, Ozan; Elmi, Abdi; Bajaj-Elliott, Mona; Taylor, Peter W; Wren, Brendan W; Dorrell, Nick

    2012-05-01

    Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

  6. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    Science.gov (United States)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by

  7. Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells.

    Science.gov (United States)

    Zhao, Chun-Hai; Chi, Zhe; Zhang, Fang; Guo, Feng-Jun; Li, Mei; Song, Wei-Bo; Chi, Zhen-Ming

    2011-05-01

    In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C(16:0), C(18:1) and C(18:2), especially C(18:1) (50.6%). Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Alterations in the Secretome of Clinically Relevant Preparations of Adipose-Derived Mesenchymal Stem Cells Cocultured with Hyaluronan

    Directory of Open Access Journals (Sweden)

    Peter Succar

    2015-01-01

    Full Text Available Osteoarthritis (OA can be a debilitating degenerative disease and is the most common form of arthritic disease. There is a general consensus that current nonsurgical therapies are insufficient for younger OA sufferers who are not candidates for knee arthroplasties. Adipose-derived mesenchymal stem cells (MSCs therapy for the treatment of OA can slow disease progression and lead to neocartilage formation. The mechanism of action is secretion driven. Current clinical preparations from adipose tissue for the treatment of OA include autologous stromal vascular fraction (SVF, SVF plus mature adipocytes, and culture-purified MSCs. Herein we have combined these human adipose-derived preparations with Hyaluronan (Hylan G-F 20: Synvisc in vitro and measured alterations in cytokine profile. SVF plus mature adipocytes showed the greatest decreased in the proinflammatory cytokines IL-1β, IFN-γ, and VEGF. MCP-1 and MIP-1α decreased substantially in the SVF preparations but not the purified MSCs. The purified MSC preparation was the only one to show increase in MIF. Overall the SVF plus mature adipocytes preparation may be most suited of all the preparations for combination with HA for the treatment of OA, based on the alterations of heavily implicated cytokines in OA disease progression. This will require further validation using in vivo models.

  9. The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Bernstein, Anke

    2016-01-01

    with CHDR. Expression of both collagens increased following addition of TGFβ. mRNA data correlated with the intensity of immunofluorescence staining. CONCLUSIONS: Paracrine effects of CHDR induce a chondrogenic phenotype in SMSC possibly mimicking joint homeostasis. Coculture approaches may lead to a better...

  10. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

    Indian Academy of Sciences (India)

    Logo of the Indian Academy of Sciences ... Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour cell spheroids with normal cells after incubation with rhTGF-1 and/or ... rhTGF-1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures.

  11. Antioxidant and bone repair properties of quercetin-functionalized hydroxyapatite: An in vitro osteoblast-osteoclast-endothelial cell co-culture study.

    Science.gov (United States)

    Forte, Lucia; Torricelli, Paola; Boanini, Elisa; Gazzano, Massimo; Rubini, Katia; Fini, Milena; Bigi, Adriana

    2016-03-01

    functionalized with different amounts of quercetin and obtain new composite materials which display both the good bioactivity of the inorganic phase and the therapeutic properties of the flavonoid. The innovative in vitro model developed in this study, which involves co-culture of osteoblast, osteoclast and endothelial cells, allows to state that the new materials exert a beneficial action onto bone repair microenvironment, stimulating osteoblast proliferation and activity, downregulating osteoclastogenesis, and supporting microangiogenetic processes necessary for new bone formation. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. CD80 and CD86 Costimulatory Molecules Differentially Regulate OT-II CD4+ T Lymphocyte Proliferation and Cytokine Response in Cocultures with Antigen-Presenting Cells Derived from Pregnant and Pseudopregnant Mice

    Directory of Open Access Journals (Sweden)

    Tomasz Maj

    2014-01-01

    Full Text Available Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c+ dendritic cells and macrophages F4/80+ and CD11b+ presenting ovalbumin (OVA were cocultured with CD4+ T cells derived from OT-II mice’s (C57BL6/J-Tg(TcraTcrb1100Mjb/J spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU, activation of these cells (flow cytometry, cytokine profile (ELISA, and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4+ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear.

  13. Fish-oil-derived n-3 PUFAs reduce inflammatory and chemotactic adipokine-mediated cross-talk between co-cultured murine splenic CD8+ T cells and adipocytes.

    Science.gov (United States)

    Monk, Jennifer M; Liddle, Danyelle M; De Boer, Anna A; Brown, Morgan J; Power, Krista A; Ma, David Wl; Robinson, Lindsay E

    2015-04-01

    Obese adipose tissue (AT) inflammation is characterized by dysregulated adipokine production and immune cell accumulation. Cluster of differentiation (CD) 8+ T cell AT infiltration represents a critical step that precedes macrophage infiltration. n-3 (ω-3) Polyunsaturated fatty acids (PUFAs) exert anti-inflammatory effects in obese AT, thereby disrupting AT inflammatory paracrine signaling. We assessed the effect of n-3 PUFAs on paracrine interactions between adipocytes and primary CD8+ T cells co-cultured at the cellular ratio observed in obese AT. C57BL/6 mice were fed either a 3% menhaden fish-oil + 7% safflower oil (FO) diet (wt:wt) or an isocaloric 10% safflower oil (wt:wt) control (CON) for 3 wk, and splenic CD8+ T cells were isolated by positive selection (via magnetic microbeads) and co-cultured with 3T3-L1 adipocytes. Co-cultures were unstimulated (cells alone), T cell receptor stimulated, or lipopolysaccharide (LPS) stimulated for 24 h. In LPS-stimulated co-cultures, FO reduced secreted protein concentrations of interleukin (IL)-6 (-42.6%), tumor necrosis factor α (-67%), macrophage inflammatory protein (MIP) 1α (-52%), MIP-1β (-62%), monocyte chemotactic protein (MCP) 1 (-23%), and MCP-3 (-19%) vs. CON, which coincided with a 74% reduction in macrophage chemotaxis toward secreted chemotaxins in LPS-stimulated FO-enriched co-culture-conditioned media. FO increased mRNA expression of the inflammatory signaling negative regulators monocyte chemoattractant 1-induced protein (Mcpip; +9.3-fold) and suppressor of cytokine signaling 3 (Socs3; +1.7-fold), whereas FO reduced activation of inflammatory transcription factors nuclear transcription factor κB (NF-κB) p65 and signal transducer and activator of transcription 3 (STAT3) by 27% and 33%, respectively. Finally, mRNA expression of the inflammasome components Caspase1 (-36.4%), Nod-like receptor family pyrin domain containing 3 (Nlrp3; -99%), and Il1b (-68.8%) were decreased by FO compared with CON (P

  14. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

    International Nuclear Information System (INIS)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

    2012-01-01

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

  15. Physiology of neuronal-glial networking

    Czech Academy of Sciences Publication Activity Database

    Verkhratsky, Alexei

    2010-01-01

    Roč. 57, č. 4 (2010), s. 332-343 ISSN 0197-0186 Institutional research plan: CEZ:AV0Z50390703 Keywords : Glia * Astrocytes * Glutamate receptors Subject RIV: FH - Neurology Impact factor: 3.601, year: 2010

  16. Dendritic cells maturated by co-culturing with HIV-1 latently infected Jurkat T cells or stimulating with AIDS-associated pathogens secrete TNF-α to reactivate HIV-1 from latency.

    Science.gov (United States)

    Ren, Xiao-Xin; Ma, Li; Sun, Wei-Wei; Kuang, Wen-Dong; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua

    2017-11-17

    Elucidation of mechanisms underlying the establishment, maintenance of and reactivation from HIV-1 latency is essential for the development of therapeutic strategies aimed at eliminating HIV-1 reservoirs. Microbial translocation, as a consequence of HIV-1-induced deterioration of host immune system, is known to result in a systemic immune activation and transient outbursts of HIV-1 viremia in chronic HIV-1 infection. How these microbes cause the robust HIV-1 reactivation remains elusive. Dendritic cells (DCs) have previously been shown to reactivate HIV-1 from latency; however, the precise role of DCs in reactivating HIV-1 from latently infected T-cell remains obscure. In this study, by using HIV-1 latently infected Jurkat T cells, we demonstrated that AIDS-associated pathogens as represented by Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) and bacterial component lipopolysaccharide (LPS) were unable to directly reactivate HIV-1 from Jurkat T cells; instead, they mature DCs to secrete TNF-α to accomplish this goal. Moreover, we found that HIV-1 latently infected Jurkat T cells could also mature DCs and enhance their TNF-α production during co-culture in a CD40-CD40L-signaling-dependent manner. This in turn led to viral reactivation from Jurkat T cells. Our results reveal how DCs help AIDS-associated pathogens to trigger HIV-1 reactivation from latency.

  17. Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.

    Science.gov (United States)

    Berger, C; Boggavarapu, N R; Menezes, J; Lalitkumar, P G L; Gemzell-Danielsson, K

    2015-04-01

    Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and

  18. Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Jin [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Liu, Qiang [Department of Hematology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yang, Jiandong [Department of Hepatobiliary Surgery, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Ren, Qinyou [Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Cao, Wei [Department of Interventional Radiology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yang, Jingyue; Yu, Zhaocai [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yu, Fang [Department of Gastrointestinal Surgery, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University, Xi' an, Shaanxi (China); Wu, Yanlan [Department of Infectious Diseases, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Shi, Hengjun [Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Liu, Wenchao [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China)

    2012-04-27

    A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.

  19. Rationally engineered synthetic coculture for improved biomass and product formation.

    Directory of Open Access Journals (Sweden)

    Suvi Santala

    Full Text Available In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.

  20. Specific CA3 neurons decode neural information of dentate granule cells evoked by paired-pulse stimulation in co-cultured networks.

    Science.gov (United States)

    Poli, Daniele; DeMarse, Thomas B; Wheeler, Bruce C; Brewer, Gregory J

    2017-07-01

    CA3 and dentate gyrus (DG) neurons are cultured in two-chamber devices on multi-electrode arrays (MEAs) and connected via micro-tunnels. In order to evoke time-locked activity, paired-pulse stimulation is applied to 22 different sites and repeated 25 times in each well in 5 MEA co-cultures and results compared to CA3-CA3 and DG-DG networks homologous controls. In these hippocampal sub-regions, we focus on the mechanisms underpinning a network's ability to decode the identity of site specific stimulation from analysis of evoked network responses using a support vector machine classifier. Our results indicate that a pool of CA3 neurons is able to reliably decode the identity of DG stimulation site information.

  1. Microfluidics co-culture systems for studying tooth innervation

    Science.gov (United States)

    Pagella, Pierfrancesco; Neto, Estrela; Jiménez-Rojo, Lucia; Lamghari, Meriem; Mitsiadis, Thimios A.

    2014-01-01

    Innervation plays a key role in the development and homeostasis of organs and tissues of the orofacial complex. Among these structures, teeth are peculiar organs as they are not innervated until later stages of development. Furthermore, the implication of neurons in tooth initiation, morphogenesis and differentiation is still controversial. Co-cultures constitute a valuable method to investigate and manipulate the interactions of nerve fibers with their target organs in a controlled and isolated environment. Conventional co-cultures between neurons and their target tissues have already been performed, but these cultures do not offer optimal conditions that are closely mimicking the in vivo situation. Indeed, specific cell populations require different culture media in order to preserve their physiological properties. In this study we evaluate the usefulness of a microfluidics system for co-culturing mouse trigeminal ganglia and developing teeth. This device allows the application of specific media for the appropriate development of both neuronal and dental tissues. The results show that mouse trigeminal ganglia and teeth survive for long culture periods in this microfluidics system, and that teeth maintain the attractive or repulsive effect on trigeminal neurites that has been observed in vivo. Neurites are repealed when co-cultured with embryonic tooth germs, while postnatal teeth exert an attractive effect to trigeminal ganglia-derived neurons. In conclusion, microfluidics system devices provide a valuable tool for studying the behavior of neurons during the development of orofacial tissues and organs, faithfully imitating the in vivo situation. PMID:25202282

  2. Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

    Science.gov (United States)

    Madrigal-Carballo, Sergio; Haas, Linda; Vestling, Martha; Krueger, Christian G; Reed, Jess D

    2016-12-01

    In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

  3. Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Kuh, Hyo-Jeong, E-mail: hkuh@catholic.ac.kr [Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of)

    2015-07-15

    Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.

  4. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution; Adduits encombrants a l'ADN dans des cocultures de cellules pulmonaires humaines exposees a une pollution atmospherique particulaire

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz [Universite Lille Nord de France - Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, MREI, Universite du Littoral Cote d' Opale, Dunkerque (France); Andre, Veronique; Le Goff, Jeremie; Sichel, Francois [GRECAN, Universite de Caen Basse-Normandie et centre Francois Baclesse, Caen (France); Roy Saint-Georges, Francoise; Mulliez, Philippe [Service de Pneumologie, Hopital Saint-Philibert, GHICL, Lille (France)

    2012-01-15

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% < 2.5{mu}.m), specific surface area (1 m{sup 2}/g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). {sup 32}P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC{sub 50} = 74.63 {mu}g/mL and L132: LC-5-0 = 75.36 {mu}g/mL). Exposure to desorbed particles (MA: C1= 61.11 {mu}g/mL and L132 : C2 = 61.71 {mu}g/mL) and B[a]P (1 {mu}M) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that

  5. Bacillus amyloliquefaciens induces production of a novel blennolide K in coculture of Setophoma terrestris.

    Science.gov (United States)

    Arora, D; Chashoo, G; Singamaneni, V; Sharma, N; Gupta, P; Jaglan, S

    2018-03-01

    The discovery of known bioactive chemical leads from microbial monocultures hinders the efficiency of drug discovery programmes. Therefore, in recent years, the use of fungal-bacterial coculture experiments has gained considerable attention due to their ability to generate new bioactive leads. In this work, fungal strain Setophoma terrestris was cocultured with Bacillus amyloliquifaciens to discover novel bioactive compounds. The bioactive methanolic coculture extract was chosen for the isolation of compounds by chromatographic methods. The isolated compounds were characterized by NMR and mass spectrometric techniques. Coculture extract has resulted in the production of five blennolides. The novel compound, blennolide K was found active against PC-3 (prostate) and MCF-7 (breast) cell lines with an IC 50 value of 3·7 ± 0·6 and 4·8 ± 0·4 μmol l -1 respectively. Furthermore, the nuclear morphology study in PC-3 cells after treatment with blennolide K, demonstrated chromatin condensation, formation of apoptotic bodies and shrinkage of cells. To our knowledge, only few studies have reported the induction of bioactive compounds by coculture having long-distance inhibition morphology. This is principally due to the low occurrences of such morphology. Our study demonstrates the impact of coculture on production of new chemical leads in drug discovery programmes. © 2017 The Society for Applied Microbiology.

  6. Expression and activation of proteases in co-cultures.

    Science.gov (United States)

    Paduch, Roman; Kandefer-Szerszeń, Martyna

    2011-01-01

    The present study concerned the expression and activation of metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system in co-cultures of human colon carcinoma cell spheroids (HT29, LS180, SW948) with human normal colon epithelium (CCD 841 CoTr), myofibroblasts (CCD-18Co) and endothelial cells (HUVEC). Additionally, the influence of monensin on the production and function of the proteases was tested. Tumor cells expressed small amounts of MMP-2, MMP-9 and uPA. Normal cells generally produced proportionally higher concentrations of these proteases (especially MMP-2, compared with significantly smaller yields of MMP-9 and significantly lower amounts of uPAR than tumors. In co-cultures of tumor spheroids with normal cell monolayers, the concentration of the proteases was equal to the sum of the enzymes produced in monocultures of both types of cells. The highest activity of uPA, measured as the reduction of the chromogenic substrate (S-2444), was detected in supernatants and lysates of endothelial cells. Interestingly, in normal cells, the higher expression of proteases, mainly uPA, measured as the level of protein concentration, was closely linked with their lower activity and inversely, in tumor cells, the low level of the expression of the enzymes correlated with their high enzymatic activity. In zymography analysis, mainly pro-MMPs were detected both in culture supernatants and cell lysates. The highest amounts of active forms of the MMPs were detected in tumor spheroids co-cultured with endothelial cells. Monensin inhibited MMPs and uPA secretion but significantly increased uPAR release, mainly from normal cells. In conclusion, during direct interactions of tumor cells with normal cells, MMPs and the uPA/uPAR system play an important role in the degradation of ECM and tumor development, but as we found, there is a reverse relationship between the concentration and the

  7. A novel technique to determine the cell type specific response within an in vitro co-culture model via multi-colour flow cytometry.

    Science.gov (United States)

    Clift, Martin J D; Fytianos, Kleanthis; Vanhecke, Dimitri; Hočevar, Sandra; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

    2017-03-27

    Determination of the cell type specific response is essential towards understanding the cellular mechanisms associated with disease states as well as assessing cell-based targeting of effective therapeutic agents. Recently, there have been increased calls for advanced in vitro multi-cellular models that provide reliable and valuable tools correlative to in vivo. In this pursuit the ability to assess the cell type specific response is imperative. Herein, we report a novel approach towards resolving each specific cell type of a multi-cellular model representing the human lung epithelial tissue barrier via multi-colour flow cytometry (FACS). We proved via ≤ five-colour FACS that the manipulation of this in vitro model allowed each cell type to be resolved with no impact upon cell viability. Subsequently, four-colour FACS verified the ability to determine the biochemical effect (e.g. oxidative stress) of each specific cell type. This technique will be vital in gaining information upon cellular mechanics when using next-level, multi-cellular in vitro strategies.

  8. Marker profiling of normal keratinocytes identifies the stroma from squamous cell carcinoma of the oral cavity as a modulatory microenvironment in co-culture

    Czech Academy of Sciences Publication Activity Database

    Lacina, L.; Dvořánková, B.; Smetana Jr, K.; Chovanec, M.; Plzák, J.; Tachezy, R.; Kideryová, L.; Kučerová, L.; Čada, Z.; Bouček, Jan; Kodet, R.; André, S.; Gabius, H. J.

    2007-01-01

    Roč. 83, 11-12 (2007), s. 837-848 ISSN 0955-3002 Grant - others:XE(XE) MRTN-CT-2005-019561; GA MZd(CZ) NR9049 Institutional research plan: CEZ:AV0Z50200510 Source of funding: R - rámcový projekt EK ; V - iné verejné zdroje Keywords : cell biology * skin * stem cells Subject RIV: EC - Immunology Impact factor: 1.468, year: 2007

  9. Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells.

    Science.gov (United States)

    Appelt-Menzel, Antje; Cubukova, Alevtina; Günther, Katharina; Edenhofer, Frank; Piontek, Jörg; Krause, Gerd; Stüber, Tanja; Walles, Heike; Neuhaus, Winfried; Metzger, Marco

    2017-04-11

    In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm 2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells in Vitro

    Science.gov (United States)

    2012-07-01

    lactic -glycolic acid (PLGA) capsules for antibiotics and rhBMP-2 delivery. Int. J. Pharm. 330:45–53. 28. Liu Y, Huse RO, de Groot K, Buser D...assay (ELISA) kit was used to test the bone cell inflammation response in the presence of bacteria . Our results suggest that, when delivered together in... bacteria are able to reach high confluence and form biofilms (38). Moreover, combined treatment with bone growth factors such as bone morphogenetic

  11. Numerical fluid-dynamic optimization of microchannel-provided porous scaffolds for the co-culture of adherent and non-adherent cells.

    Science.gov (United States)

    Cantini, Marco; Fiore, Gianfranco B; Redaelli, Alberto; Soncini, Monica

    2009-03-01

    Computational fluid dynamic (CFD) techniques were used to optimize the microenvironment inside scaffolds for hematopoietic stem cell (HSC) culture in a perfusion bioreactor. These matrices are meant to be seeded with adherent bone marrow stromal cells and then co-cultivated with HSCs; the scaffold micro-architecture and the fluid-dynamic conditions have to be optimized to avoid non-adherent stem cells being dragged away while ensuring adequate nutrient supply. The insertion of longitudinal microchannels was tested as a tool to improve perfusion in a homogeneous porous scaffold. Models of microchannel-provided scaffolds, characterized by different values of geometric parameters concerning pores and channels, were built, and numerical fluid-dynamic and oxygen-transfer analyses were carried out. The results of the computations indicated that the microchannels created preferential paths for culture medium flow, causing low shear stresses and drag forces within the pores; meanwhile, they improved oxygen delivery by forcing its penetration into the scaffold bulk. In particular, an 85% porous, 3-mm-thick scaffold with 175-microm-diameter pores was considered; at a constant average drag force guaranteeing stem cell suspension inside this porous bulk, the addition of approximately 260-microm-diameter, 700-microm-spaced channels resulted in 34% higher oxygen partial pressure at the exit (approximately 135 vs 101 mmHg), maintaining a wall shear stress median value of approximately 0.14 mPa. The present work demonstrates the capacity of microchannel-provided scaffolds to ensure suitable conditions for HSC culture and shows that CFD methods are a valuable tool to retrieve significant clues for the design of the culture environment.

  12. Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar-endothelial co-cultures

    DEFF Research Database (Denmark)

    Cao, Yi; Roursgaard, Martin; Jacobsen, Nicklas Raun

    2016-01-01

    Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We...... measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549...

  13. Neural Transplantation Model Using Integration Co-culture Chamber

    Science.gov (United States)

    Shimba, Kenta; Saito, Atsushi; Takeuchi, Akimasa; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

    Regenerative medicine is a promising therapy for injuries and diseases of the central nervous system (CNS). Implantation of stem cell-derived neurons into the recipient tissue is one of the key processes of the therapy. How the implanted cells establish functional connections with the intact neurons, and whether the established connections are maintained stably for a long time, remain unknown. Here, we report a novel co-culture device for visualizing interconnections between primary and differentiated neuronal cultures, and long-term monitoring of neuronal activity. A circular micro-chamber surrounded by another chamber is aligned on a microelectrode array (MEA). These chambers are interconnected through 36 micro-tunnels. Stem cell-derived neurons were cultured in the inner circular chamber, and primary neurons taken from mouse cortices were cultured in the surrounding chamber. Neurites outgrew into the micro-tunnels from both primary and differentiated neurons. The immunofluorescence images indicate that synaptic connections are formed between them. Propagation of electrical activity was observed 6 days after starting co-culture. More than half of the spontaneous activity was initiated from primary neurons, and probability of activity propagation to the stem cell-derived neurons gradually increased with culture days. These results suggest that our device is feasible for long-term monitoring of interaction between stem cell-derived cells and the recipient tissue.

  14. Self-sustaining, solar-driven bioelectricity generation in micro-sized microbial fuel cell using co-culture of heterotrophic and photosynthetic bacteria

    Science.gov (United States)

    Liu, Lin; Choi, Seokheun

    2017-04-01

    Among many energy harvesting techniques with great potential, microbial fuel cell (MFC) technology is arguably the most underdeveloped. Even so, excitement is building, as microorganisms can harvest electrical power from any biodegradable organic source (e.g. wastewater) that is readily available in resource-limited settings. Nevertheless, the requirement for endless introduction of organic matter imposes a limiting factor to this technology, demanding an active feeding system and additional power. Here, we demonstrated self-sustaining bioelectricity generation from a microliter-scale microbial fuel cell (MFC) by using the syntrophic interaction between heterotrophic exoelectrogenic bacteria and phototrophs. The MFC continuously generated light-responsive electricity from the heterotrophic bacterial metabolic respiration with the organic substrates produced by photosynthetic bacteria. Without additional organic fuel, the mixed culture in a 90-μL-chamber MFC generated self-sustained current for more than 13 days, while the heterotrophic culture produced current that decreased dramatically within a few hours. The current from the mixed culture was about 70 times greater than that of the device with only photosynthetic bacteria. The miniaturization provided a short start-up time, a well-controlled environment, and small internal resistance. Those advantages will become the general design platform for micropower generation.

  15. Co-Cultured Continuously Bioluminescent Human Cells as Bioreporters for the Detection of Prodrug Therapeutic Impact Pre- and Post-Metabolism

    Directory of Open Access Journals (Sweden)

    Tingting Xu

    2017-12-01

    Full Text Available Modern drug discovery workflows require assay systems capable of replicating the complex interactions of multiple tissue types, but that can still function under high throughput conditions. In this work, we evaluate the use of substrate-free autobioluminescence in human cell lines to support the performance of these assays with reduced economical and logistical restrictions relative to substrate-requiring bioluminescent reporter systems. The use of autobioluminescence was found to support assay functionality similar to existing luciferase reporter targets. The autobioluminescent assay format was observed to correlate strongly with general metabolic activity markers such as ATP content and the presence of reactive oxygen species, but not with secondary markers such as glutathione depletion. At the transcriptional level, autobioluminescent dynamics were most closely associated with expression of the CYP1A1 phase I detoxification pathway. These results suggest constitutively autobioluminescent cells can function as general metabolic activity bioreporters, while pairing expression of the autobioluminescent phenotype to detoxification pathway specific promoters could create more specific sensor systems.

  16. [Research progress of co-culture system for constructing vascularized tissue engineered bone].

    Science.gov (United States)

    Fu, Weili; Xiang, Zhou

    2014-02-01

    To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

  17. Co-culture systems and technologies: taking synthetic biology to the next level.

    Science.gov (United States)

    Goers, Lisa; Freemont, Paul; Polizzi, Karen M

    2014-07-06

    Co-culture techniques find myriad applications in biology for studying natural or synthetic interactions between cell populations. Such techniques are of great importance in synthetic biology, as multi-species cell consortia and other natural or synthetic ecology systems are widely seen to hold enormous potential for foundational research as well as novel industrial, medical and environmental applications with many proof-of-principle studies in recent years. What is needed for co-cultures to fulfil their potential? Cell-cell interactions in co-cultures are strongly influenced by the extracellular environment, which is determined by the experimental set-up, which therefore needs to be given careful consideration. An overview of existing experimental and theoretical co-culture set-ups in synthetic biology and adjacent fields is given here, and challenges and opportunities involved in such experiments are discussed. Greater focus on foundational technology developments for co-cultures is needed for many synthetic biology systems to realize their potential in both applications and answering biological questions. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  18. Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies.

    Science.gov (United States)

    Li, Guanglei; Simon, Melissa J; Cancel, Limary M; Shi, Zhong-Dong; Ji, Xinying; Tarbell, John M; Morrison, Barclay; Fu, Bingmei M

    2010-08-01

    The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L (p)), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L (p) and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

  19. Co-culture systems-based strategies for articular cartilage tissue engineering.

    Science.gov (United States)

    Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

    2018-03-01

    Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

  20. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

    Indian Academy of Sciences (India)

    We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the ...

  1. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

    Indian Academy of Sciences (India)

    Prakash

    2009-12-09

    Dec 9, 2009 ... 2Department of Comparative Anatomy and Anthropology, Institute of Biology, Maria Curie-Skłodowska University, ... in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human ..... expression of HSP72 and MRP in human colon tumour cells.

  2. Streptococcus pneumoniae cocultured with fibroblasts enhances both interferon production and cytotoxic activity by lymphocytes.

    OpenAIRE

    Weigent, D A; Baron, S; Stanton, G J

    1985-01-01

    Cell-mediated cytotoxicity against normal human fibroblasts was dependent on treatment of the fibroblasts with Streptococcus pneumoniae. Both spontaneous and interferon (IFN)-enhanced lymphocytes killed human foreskin (HFS) or skin muscle cells cocultured with S. pneumoniae five- to eightfold more than control nontreated cells. Based on Percoll gradient centrifugation, the cytotoxic effector cell migrated like a large granular lymphocyte. The human IFN produced from mixtures of HFS cells, lym...

  3. Design of biomimetic cellular scaffolds for co-culture system and their application.

    Science.gov (United States)

    Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

    2017-01-01

    The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell-cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment.

  4. A TRACER 3D Co-Culture tumour model for head and neck cancer.

    Science.gov (United States)

    Young, Miki; Rodenhizer, Darren; Dean, Teresa; D'Arcangelo, Elisa; Xu, Bin; Ailles, Laurie; McGuigan, Alison P

    2018-05-01

    Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment and have been shown to play an important role in the progression of cancer. To probe these tumour-stroma interactions, we incorporated CAFs derived from head and neck cancer patients and squamous carcinoma cells of the hypopharynx (FaDu) into the Tissue Roll for the Analysis of Cellular Environment and Response (TRACER) platform to establish a co-culture platform that simulates the CAF-tumour microenvironmental interactions in head and neck tumours. TRACER culture involves infiltrating cells into a thin fibrous scaffold and then rolling the resulting biocomposite around a mandrel to generate a 3D and layered structure. Patterning the fibrous scaffold biocomposite during fabrication enables control over the specific location of different cell populations in the rolled configuration. Here, we optimized the seeding densities and configurations of the CAF and FaDu cell tissue sections to enable a robust 3D co-culture system under normoxic conditions. Co-culture of CAFs with FaDu cells produced negligible effects on radiation resistance, but did produce increases in proliferation rate and invasive cell migration at 24 and 48 h of culture. Our study provides the basis for use of our in vitro co-culture TRACER model to investigate the tumour-stroma interactions, and to bridge the translational gap between preclinical and clinical studies. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Tumor-suppressor effects of chemical functional groups in an in vitro co-culture system

    Science.gov (United States)

    Xu, Su-Ju; Cui, Fu-Zhai; Kong, Xiang-Dong

    2014-06-01

    Liver normal cells and cancer cells co-cultured on surfaces modified by different chemical functional groups, including mercapto (-SH), hydroxyl (-OH) and methyl (-CH3) groups. The results showed that different cells exhibited changes in response to different surfaces. Normal cells on -SH surface exhibited the smallest contact area with mostly rounded morphology, which led to the death of cancer cells, while cancer cells could not grow on -CH3 groups, which also died. In the co-culture system, the -CH3 group exhibited its unique effect that could trigger the death of cancer cells and had no effects on normal cells. Our findings provide useful information on strategies for the design of efficient and safe regenerative medicine materials.

  6. Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

    Science.gov (United States)

    Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

    2017-08-07

    The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase ( p culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

  7. Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes

    Directory of Open Access Journals (Sweden)

    Robert Krencik

    2017-12-01

    Full Text Available Summary: Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC-derived astrocytes (hAstros combined with neurons generated from hPSC-derived neural stem cells (hNeurons or directly induced via Neurogenin 2 overexpression (iNeurons. Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations. : In this article, Krencik and colleagues show that high-density cocultures of pre-differentiated human astrocytes with induced neurons, from pluripotent stem cells, elicit mature characteristics by 3–5 weeks. This provides a faster and more defined alternative method to organoid cultures for investigating human neural circuit function. Keywords: human pluripotent stem cells, neurons, astrocytes, synapses, coculture, three-dimensional spheres, organoids, disease modeling

  8. Use of a Coculture To Enable Current Production by Geobacter sulfurreducens

    KAUST Repository

    Qu, Y.

    2012-02-17

    Microbial fuel cells often produce more electrical power with mixed cultures than with pure cultures. Here, we show that a coculture of a nonexoelectrogen (Escherichia coli) and Geobacter sulfurreducens improved system performance relative to that of a pure culture of the exoelectrogen due to the consumption of oxygen leaking into the reactor.

  9. Changes in growth and survival of Bifidobacterium by coculture with Propionibacterium in soy milk, cow's milk, and modified MRS medium.

    Science.gov (United States)

    Wu, Qian Qian; You, Hyun Ju; Ahn, Hyung Jin; Kwon, Bin; Ji, Geun Eog

    2012-06-15

    Bifidobacterium adolescentis Int57 (Int57) and Propionibacterium freudenreichii subsp. shermanii ATCC 13673 (ATCC 13673) were grown either in coculture or as pure cultures in different media, such as cow's milk, soybean milk, and modified MRS medium. The viable cell counts of bacteria, changes in pH, concentrations of organic acids, and contents of various sugars were analyzed during incubation up to 7days. In soy milk, the survival of cocultured Int57 was six times higher than the monocultured cells, and ATCC 13673 cocultured with Int57 consumed 69.4% of lactic acid produced by Int57 at the end of fermentation. In cow's milk, coculture with ATCC 13673 increased the growth of Int57 from 24h until 120h by approximately tenfold and did not affect the survival of Int57 cells. After 96h of fermentation of modified MRS, the survival of ATCC 13673 cells cocultured with Int57 increased by 3.2- to 7.4-folds as compared with ATCC 13673 monoculture, whereas the growth of Int57 cells was unaffected. The growth and metabolic patterns of two strains during coculture showed noticeable differences between food grade media and laboratory media. The consumption of stachyose in soy milk during coculture of Int57 with ATCC 13673 was increased by more than twice compared with Int57 monoculture, and completed within 24h. The combinational use of Bifidobacterium and Propionibacterium could be applied to the development of fermented milk or soy milk products. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

    Directory of Open Access Journals (Sweden)

    Mahmoud Heidari

    2011-01-01

    Full Text Available Background: The aim of this study was to evaluate fibroblast co-culture on in vitro maturation andfertilization of prepubertal mouse preantral follicles.Materials and Methods: The ovaries of 12-14 day old mice were dissected and 120-150 μmintact preantral follicles with one or two layers of granulosa cells, and round oocytes were culturedindividually in α-minimal essential medium (α-MEM supplemented with 5% fetal bovine serum(FBS, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin, seleniummix, 100 μg/ml penicillin and 50 μg/ml streptomycin as base medium for 12 days. A total number of226 follicules were cultured under two conditions: i base medium as control group (n=113; ii basemedium co-cultured with mouse embryonic fibroblast (MEF (n=113. Follicular diameters, alone,in addition to other factors were analyzed by student’s t-test and chi-square test, respectively.Results: The co-culture group showed significant differences (p<0.05 in growth rate (days 4, 6 and8 of the culture period and survival rate. However, there was no significant difference in antrumformation, ovulation rate and embryonic development of released oocytes. There were significantdifferences (p<0.05 in the estradiol and progesterone secretion at all days between the co-cultureand control groups.Conclusion: Fibroblast co-culture increased survival rate and steroid production of preantralfollicles by promoting granulosa cell proliferation.

  11. Immobilized anaerobic fermentation for bio-fuel production by Clostridium co-culture.

    Science.gov (United States)

    Xu, Lei; Tschirner, Ulrike

    2014-08-01

    Clostridium thermocellum/Clostridium thermolacticum co-culture fermentation has been shown to be a promising way of producing ethanol from several carbohydrates. In this research, immobilization techniques using sodium alginate and alkali pretreatment were successfully applied on this co-culture to improve the bio-ethanol fermentation performance during consolidated bio-processing (CBP). The ethanol yield obtained increased by over 60 % (as a percentage of the theoretical maximum) as compared to free cell fermentation. For cellobiose under optimized conditions, the ethanol yields were approaching about 85 % of the theoretical efficiency. To examine the feasibility of this immobilization co-culture on lignocellulosic biomass conversion, untreated and pretreated aspen biomasses were also used for fermentation experiments. The immobilized co-culture shows clear benefits in bio-ethanol production in the CBP process using pretreated aspen. With a 3-h, 9 % NaOH pretreatment, the aspen powder fermentation yields approached 78 % of the maximum theoretical efficiency, which is almost twice the yield of the untreated aspen fermentation.

  12. [Experimental study on co-culture of human fibroblasts on decellularized Achilles tendon].

    Science.gov (United States)

    Wang, Zhibing; Zhang, Xia; Guo, Xinyu; Qin, Chuan

    2013-07-01

    To investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits (5-month-old; weighing, 4-5 kg). The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate (SDS), and then gross observation, histological examination, and scanning electron microscope (SEM) observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. After decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young's elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group (P Achilles tendons group and cell-scaffold composite group (P > 0.05). There was no significant difference in elongation at break among 3 groups (P > 0.05). The decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.

  13. Modelling the effects of PSII inhibitor pulse exposure on two algae in co-culture.

    Science.gov (United States)

    Copin, Pierre-Jean; Chèvre, Nathalie

    2018-03-01

    A weakness of standard testing procedures is that they do not consider interactions between organisms, and they focus only on single species. Furthermore, these procedures do not take into account pulse exposure. However, pulse exposure is of particular importance because in streams, after crop application and during and after precipitation, herbicide concentrations fluctuate widely and can exceed the Annual Average Environmental Quality Standards (AA-EQS), which aim to protect the aquatic environment. The sensitivity of the algae Scenedesmus vacuolatus and Pseudokirchneriella subcapitata in a co-culture exposed to pulses is thus analysed in this study. As a first step, the growths of the algae in co-culture are investigated. For initial cell densities fixed, respectively, to 100,000 and 50,000 cells/mL, the growth of each alga is exponential over at least 48 h. S. vacuolatus seems to influence the growth of P. subcapitata negatively. Allelopathy is a possible explanation for this growth inhibition. The toxicity of the herbicide isoproturon is later tested on the algae S. vacuolatus and P. subcapitata cultured alone and in the co-culture. Despite the supplementary stress on the algae in the co-culture competing for nutrients, the toxicity of the herbicide is lower for the two algae when they are in the co-culture than when they are in separated culture. A model is adapted and used to predict the cell-density inhibition on the alga S. vacuolatus in the co-culture with the alga P. subcapitata exposed to a pulse concentration of isoproturon. Four laboratory experiments are performed to validate the model. The comparison between the laboratory and the modelled effects shows good agreement. The differences can be considered minor most of time. For future studies, it is important to ensure that the cell count is precise, as it is used to determine the parameters of the model. The differences can be also induced by the fact that the cell number of the alga P

  14. Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics

    Science.gov (United States)

    Yao, Lu; Zhu, Li-Ping; Xu, Xiao-Yan; Tan, Ling-Ling; Sadilek, Martin; Fan, Huan; Hu, Bo; Shen, Xiao-Ting; Yang, Jie; Qiao, Bin; Yang, Song

    2016-09-01

    Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-β-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.

  15. Transport Study of Egg-Derived Antihypertensive Peptides (LKP and IQW) Using Caco-2 and HT29 Coculture Monolayers.

    Science.gov (United States)

    Xu, Qingbiao; Fan, Hongbing; Yu, Wenlin; Hong, Hui; Wu, Jianping

    2017-08-30

    The objective of this study was to investigate the mechanisms of the transport of antihypertensive tripeptides LKP (Leu-Lys-Pro) and IQW (Ile-Gln-Trp) derived from egg white using a coculture system of Caco-2 and HT29 cell monolayers. The results revealed that LKP and IQW have no cytotoxicity to the cell viability after 2 h incubation, could be transported intact across coculture monolayers (apparent permeability coefficient: (18.11 ± 1.57) × 10 -8 and (13.21 ± 1.12) × 10 -8 cm/s, respectively), and were resistant to peptidase secreted by enterocytes. In addition, the transports were significantly inhibited by dipeptide Gly-Pro (P Caco-2/HT29 coculture monolayers.

  16. Development of On-chip Coculture System for Cytotoxicity Test Using Caco-2 and Hep G2

    Science.gov (United States)

    Kimura, Hiroshi; Nakayama, Hidenari; Yamamoto, Takatoki; Sakai, Yasuyuki; Fujii, Teruo

    We developed a chip-based coculture system for cytotoxicity test, as our continuous effort to develop a multi-functional micro culture device realized by integration of fluidic control. The culture zone in the device was divided into two compartments separated by a microporous membrane through which substances in culture medium can freely come-and-go to induce the mutual interactions between the cells cultured at each compartment. In this work, it was examined that 1) coculture and 2) cytotoxicity model through oral intake, using Caco-2 and Hep G2 cell as a model cell of small intestine and liver respectively. As a result of test 1), Hep G2 cells cocultured with Caco-2 show same albumin secretion activity as the one not cocultured with Caco-2 cells. As a result of test 2), The cytotoxicity of caffeine and paraquat on Hep G2 cells was successfully measured with and without association of a selective chemical barrier function of Caco-2 cells.

  17. Naringenin-responsive riboswitch-based fluorescent biosensor module for Escherichia coli co-cultures.

    Science.gov (United States)

    Xiu, Yu; Jang, Sungho; Jones, J Andrew; Zill, Nicholas A; Linhardt, Robert J; Yuan, Qipeng; Jung, Gyoo Yeol; Koffas, Mattheos A G

    2017-10-01

    The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. Cracking Streptococcus thermophilus to stimulate the growth of the probiotic Lactobacillus casei in co-culture.

    Science.gov (United States)

    Ma, Chengjie; Ma, Aimin; Gong, Guangyu; Liu, Zhenmin; Wu, Zhengjun; Guo, Benheng; Chen, Zhengjun

    2015-10-01

    Lactobacillus casei, a probiotic, and Streptococcus thermophilus, a fast acidifying lactic acid bacterial strain, are both used in the food industry. The aim of this study was to investigate the interaction between L. casei and S. thermophilus in the presence or absence of S. thermophilus-specific bacteriophage during milk fermentation. The acidification capability of L. casei co-cultured with S. thermophilus was significantly higher than that observed for L. casei or S. thermophilus cultured alone. However, the probiotic content (i.e., L. casei cell viability) was low. The fastest acidification and the highest viable L. casei cell count were observed in co-cultures of L. casei and S. thermophilus with S. thermophilus phage. In these co-cultures, S. thermophilus compensated for the slow acid production of L. casei in the early exponential growth phase. Thereafter, phage-induced lysis of the S. thermophilus cells eliminated the competition for nutrients, allowing L. casei to grow well. Additionally, the ruptured S. thermophilus cells released intracellular factors, which further promoted the growth and function of the probiotic bacteria. Crude cellular extract isolated from S. thermophilus also significantly accelerated the growth and propagation of L. casei, supporting the stimulatory role of the phage on this micro-ecosystem. Copyright © 2015. Published by Elsevier B.V.

  19. Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation

    Science.gov (United States)

    Correia, Clara R.; Pirraco, Rogério P.; Cerqueira, Mariana T.; Marques, Alexandra P.; Reis, Rui L.; Mano, João F.

    2016-02-01

    A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

  20. Butyrate attenuates lipolysis in adipocytes co-cultured with macrophages through non-prostaglandin E2-mediated and prostaglandin E2-mediated pathways.

    Science.gov (United States)

    Ohira, Hideo; Tsutsui, Wao; Mamoto, Rie; Yamaguchi, Sayaka; Nishida, Masako; Ito, Miki; Fujioka, Yoshio

    2016-12-09

    Interactions between adipocytes and macrophages are associated with metabolic disorders. Production of pro-inflammatory mediators and the release of free fatty acids (FFAs) increase when these cells are co-cultured; butyrate significantly diminishes these effects by suppressing both the macrophage inflammatory and adipocyte lipolysis pathways. Butyrate is known to up-regulate the expression of prostaglandin E2 (PGE2). Therefore, we hypothesized that PGE2 is associated with the suppression of lipolysis by butyrate in co-culture. Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the release of PGE2 into the medium and on lipolysis in adipocytes. To elucidate the underlying mechanism, we examined the effects of butyrate on cyclooxygenase-2 (COX2) and phospholipase A2 (PLA2) in co-cultured cells, and cyclic adenine monophosphate (cAMP) and protein kinase A type 1-α regulatory subunit (PRKAR1A) in co-cultured adipocytes. Silent interfering (si)RNA targeting of G-protein-coupled receptor (GPR)41 and 109A was employed to examine the effect on lipolysis in TNF-α-stimulated adipocytes. Co-culture increased PGE2 release into the medium, compared with cells cultured separately. Butyrate significantly increased PGE2 production. Co-culture elevated COX2 expression in macrophages and adipocytes, and butyrate further enhanced this effect. Co-culture enhanced cytosolic PLA2 activity in macrophages, which was further enhanced by butyrate. As for lipolysis, co-culture increased the release of FFAs and free glycerol into the medium, whereas butyrate (and to a lesser extent, PGE2) suppressed FFAs and free glycerol release. An inhibition study using a prostaglandin E receptor 3-selective antagonist suggested that approximately 40% of the suppressive effect of butyrate depends on the PGE2-mediated pathway, whereas 60% depends on a non-PGE2-mediated pathway. Co-culture increased c

  1. Microalgae harvesting via co-culture with filamentous fungus

    Science.gov (United States)

    Gultom, Sarman Oktovianus

    Microalgae harvesting is a labor- and energy-intensive process. For instance, classical harvesting technologies such as chemical addition and mechanical separation are economically prohibiting for biofuel production. Newer approaches to harvest microalgae have been developed in order to decrease costs. Among these new methods, fungal co-pelletization seems to be a promising technology. By co-culturing filamentous fungi with microalgae, it is possible to form pellets, which can easily be separated. In this study, different parameters for the cultivation of filamentous fungus (Aspergillus niger) and microalgae (Chlorella vulgaris) to efficiently form cell pellets were evaluated under heterotrophic and phototrophic conditions, including organic carbon source (glucose, glycerol and sodium acetate) concentration, pH, initial concentration of fungal spores, initial concentration of microalgal cells, concentration of ionic strength (Calcium and Magnesium) and concentration of salinity (NaCl). In addition, zeta-potential measurements were carried out in order to get a better understanding of the mechanism of attraction. It was found that 2 g/L of glucose, a fungus to microalgae ratio of 1:300, and uncontrolled pH (around 7) are the best culturing conditions for co-pelletization. Under these conditions, it was possible to achieve a high harvesting performance (>90%). In addition, it was observed that most pellets formed in the co-culture were spherical with an average diameter of 3.5 mm and in concentrations of about 5 pellets per mL of culture media. Under phototrophic conditions, co-pelletization required the addition of glucose as organic carbon source to sustain the growth of fungi and to allow the harvesting of microalgae. Zeta-potential measurements indicated that (i) both microalgae and fungi have low zeta-potential values regardless of the pH on the bulk (i.e. <-10 mV) (ii) fungi can have a positive electric charge at low pH (ie. pH=3). These values suggest that it

  2. Arginine does not exacerbate markers of inflammation in cocultures of human enterocytes and leukocytes

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Negrier, I.; Neveux, N.

    2007-01-01

    the catabolism of serine, asparagine, and lysine, and reduced glutamine catabolism. Addition of arginine increased ornithine formation and moderately reduced transepithelial transport of methionine and other amino acids. Hence, arginine supplementation does not interfere with inflammation-associated cross......Enteral arginine supplementation in the critically ill has become a matter of controversy. In this study, we investigated effects of the addition of 0.4 and 1.2 mmol/L arginine in a coculture model on markers of inflammation, enterocyte layer integrity, and amino acid transport. In this model...... with arginine did not affect epithelial integrity, production of any of the cytokines investigated, or the amount of nitric oxide. The amino acid used primarily by nonstimulated intestinal epithelial cells cocultured with leukocytes was glutamine. Activation of IEC with bacteria significantly enhanced...

  3. Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4.

    Science.gov (United States)

    Mori, Y

    1990-01-01

    Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.

  4. Neuron-oligodendrocyte myelination co-culture derived from embryonic rat spinal cord and cerebral cortex.

    Science.gov (United States)

    Pang, Yi; Zheng, Baoying; Kimberly, Simpson L; Cai, Zhengwei; Rhodes, Philip G; Lin, Rick C S

    2012-01-01

    An in vitro myelination model derived from rat central nervous system (CNS) remains to be established. Here, we describe a simple and reproducible myelination culture method using dissociated neuron-oligodendrocyte (OL) co-cultures from either the embryonic day 16 (E16) rat spinal cord or cerebral cortex. The dissociated cells are plated directly on poly-L-lysine-coated cover slips and maintained in a modified myelination medium that supports both OL and neuron differentiation. The spinal cord derived OL progenitor cells develop quickly into myelin basic protein (MBP)+ mature OLs and start to myelinate axons around 17 days in vitro (DIV17). Myelination reaches its peak around six weeks (DIV40) and the typical nodes of Ranvier are revealed by paranodal proteins Caspr and juxaparanodal protein Kv1.2 immunoreactivity. Electron microscopy (EM) shows typical myelination cytoarchitecture and synaptic organization. In contrast, the cortical-derived co-culture requires triiodothyronine (T3) in the culture medium for myelination. Finally, either hypomyelination and/or demyelination can be induced by exposing proinflammatory cytokines or demyelinating agents to the co-culture, suggesting the feasibility of this modified in vitro myelination model for myelin-deficit investigation.

  5. Co-culturing Effects of Coexisting Bacteria on Wood Degradation by Trametes versicolor.

    Science.gov (United States)

    Kamei, Ichiro

    2017-01-01

    White-rot fungi are the main decomposers of wood cell-wall polymer in forest ecosystems. Little is known, however, about the interactions between white-rot fungi and other coexisting microorganisms in decayed wood. A white-rot fungus, Trametes versicolor strain TN6F, was isolated from a fruit body, and 44 strains of coexisting cultivable bacteria were isolated from its substrate, natural white rot-decayed wood. The effects of these bacteria on fungal growth were examined by an in vitro confrontation growth assay. Among the isolates, nine bacterial strains inhibited the growth of strain TN6F, while 35 strains did not affect the growth of TN6F. However, when co-cultured with strain TN6F on wood powder, many bacterial strains promoted the weight loss of the substrate. A subsequent chemical composition analysis showed that co-culturing accelerated delignification. Higher laccase activity was detected when strain TN6F was co-cultured on wood powder medium with bacterial strains TN6W-26 or TN6W-27. These results indicate that some bacterial strains might promote wood degradation.

  6. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: Comparison with conventional monocultures

    Directory of Open Access Journals (Sweden)

    Stauber Roland

    2011-01-01

    Full Text Available Abstract Background To date silica nanoparticles (SNPs play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities. They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures. Methods Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release, and TER (Transepithelial Electrical Resistance measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release and apoptosis markers were investigated. Results Regarding toxic effects (viability, membrane integrity, TER the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number

  7. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2015-12-01

    Full Text Available BACKGROUND Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. METHODS In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. RESULTS Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1- fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity CONCLUSIONS Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  8. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2014-08-01

    Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  9. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    Science.gov (United States)

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  10. Effects of platelet rich plasma and chondrocyte co-culture on MSC chondrogenesis, hypertrophy and pathological responses

    OpenAIRE

    Ramezanifard, Rouhallah; Kabiri, Mahboubeh; Hanaee Ahvaz, Hana

    2017-01-01

    Regarding the inadequate healing capability of cartilage tissue, cell-based therapy is making the future of cartilage repair and regeneration. Mesenchymal stem cells (MSC) have shown great promise in cartilage regeneration. However, a yet-unresolved issue is the emergence of hypertrophic and pathologic markers during in vitro MSC chondrogenesis. Articular chondrocytes (AC) can suppress the undesired hypertrophy when co-cultured with MSC. On the other hand, platelet rich plasma (PRP), is consi...

  11. A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity.

    Science.gov (United States)

    Pamies, David; Barreras, Paula; Block, Katharina; Makri, Georgia; Kumar, Anupama; Wiersma, Daphne; Smirnova, Lenna; Zang, Ce; Bressler, Joseph; Christian, Kimberly M; Harris, Georgina; Ming, Guo-Li; Berlinicke, Cindy J; Kyro, Kelly; Song, Hongjun; Pardo, Carlos A; Hartung, Thomas; Hogberg, Helena T

    2017-01-01

    Human in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology.

  12. Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication

    Directory of Open Access Journals (Sweden)

    Trindade Edvaldo S

    2009-08-01

    Full Text Available Abstract Background Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM, used as an immune modulator, has been recommended for patients with depressed immune systems. Previous studies in mice have demonstrated that the CHM activates macrophages, induces an increase in the number of leukocytes and improves the murine response against Sarcoma-180. Methods Here we studied the interaction of mouse lymph node lymphocytes, co-cultured in vitro with macrophages in the presence or absence of the CHM, with B16F10 melanoma cells. Results Lymphocytes co-cultured with macrophages in the presence of the CHM had greater anti-melanoma activity, reducing melanoma cell density and increasing the number of lysed tumor cells. There was also a higher proportion of activated (CD25+ lymphocytes with increased viability. Overall, lymphocytes activated by treatment destroyed growing cancer cells more effectively than control lymphocytes. Conclusion Co-culture of macrophages with lymphocytes in the presence of the CHM enhanced the anti-cancer performance of lymphocytes against a very aggressive lineage of melanoma cells. These results suggest that non-toxic therapies using CHMs are a promising alternative approach to the treatment of melanomas. In addition, they are attractive combination-therapy candidates, which may enhance the efficacy of conventional medicines by improving the immune response against tumor cells.

  13. Effects of the probiotic Enterococcus faecium NCIMB 10415 on selected lactic acid bacteria and enterobacteria in co-culture.

    Science.gov (United States)

    Starke, I C; Zentek, J; Vahjen, W

    2015-01-01

    Enterococcus faecium NCIMB 10415 is used as a probiotic for piglets and has been shown to modify the porcine intestinal microbiota. However, the mode of action of this probiotic modification is still unclear. One possible explanation is the direct growth inhibiting or stimulating effect of the probiotic on other indigenous bacteria. Therefore, the aim of the present study was to examine the growth interactions of the probiotic with different indigenous porcine bacteria in vitro. Reference strains were cultivated with the probiotic E. faecium strain NCIMB10415 (SF68) in a checkerboard assay with 102 to 105 cells/ml inoculum per strain. Growth kinetics were recorded for 8 h and used to determine specific growth of the co-cultures. Additionally, total DNA was extracted from the co-cultures at the end of the incubation to verify which strain in the co-culture was affected. Co-cultivation with eight Enterococcus spp. tester strains showed strain-specific growth differences. Three of four E. faecium strains were not influenced by the probiotic strain. PCR results showed reduced growth of the probiotic strain in co-culture with E. faecium DSM 6177. Three of four Enterococcus faecalis strains showed reduced specific growth in co-culture with the probiotic strain. However, E. faecalis DSM 20478 impaired growth of the probiotic E. faecium strain. The growth of Lactobacillus johnsonii DSM 10533 and Lactobacillus reuteri DSM 20016 was enhanced in co-culture with the probiotic strain, but co-cultivations with Lactobacillus mucosae DSM13345 or Lactobacillus amylovorus DSM10533 showed no differences. Co-cultures with the probiotic E. faecium showed no impact on the growth rate of four different enterobacterial reference strains (2 strains of Salmonella enterica and 2 strains of Escherichia coli), but PCR results showed reduced cell numbers for a pathogenic E. coli isolate at higher concentration of the probiotic strain. As the in vitro effect of the probiotic E. faecium on

  14. Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells.

    Science.gov (United States)

    Alais, Sandrine; Dutartre, Hélène; Mahieux, Renaud

    2017-01-01

    Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.

  15. Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits

    Energy Technology Data Exchange (ETDEWEB)

    Powell, Joshua D.; Hess, Becky M.; Hutchison, Janine R.; Straub, Tim M.

    2015-05-01

    We report the construction of an in vitro three dimensional (3D) co-culture platform consisting of differentiated lung epithelial cells and monocytes from New Zealand white rabbits. Rabbit lung epithelial cells were successfully grown at air-liquid interface, produced mucus, and expressed both sialic acid alpha-2,3 and alpha-2,6. Blood-derived CD14+ monocytes were deposited above the epithelial layer resulting in the differentiation of a subset of monocytes into CD11c+ cells within the co-culture. These proof-of-concept findings provide a convenient means to comparatively study in vitro versus in vivo rabbit lung responses as they relate to inhalation or lung-challenge studies.

  16. A 3D co-culture microtissue model of the human placenta for nanotoxicity assessment

    DEFF Research Database (Denmark)

    Muoth, Carina; Wichser, Adrian; Monopoli, Marco

    2016-01-01

    in 3D than in 2D cell cultures, which indicates an enhanced differentiation of trophoblasts grown on 3D MTs. NP toxicity assessment revealed that cadmium telluride (CdTe) and copper oxide (CuO) NPs but not titanium dioxide (TiO2) NPs decreased MT viability and reduced the release of hCG. NP acute...... toxicity was significantly reduced in 3D co-culture MTs compared to 2D monocultures. Taken together, 3D placental MTs provide a new and promising model for the fast generation of tissue-relevant acute NP toxicity data, which are indispensable for the safe development of NPs for industrial, commercial...

  17. Effects of silver nanoparticles and ions on a co-culture model for the gastrointestinal epithelium.

    Science.gov (United States)

    Georgantzopoulou, Anastasia; Serchi, Tommaso; Cambier, Sébastien; Leclercq, Céline C; Renaut, Jenny; Shao, Jia; Kruszewski, Marcin; Lentzen, Esther; Grysan, Patrick; Eswara, Santhana; Audinot, Jean-Nicolas; Contal, Servane; Ziebel, Johanna; Guignard, Cédric; Hoffmann, Lucien; Murk, AlberTinka J; Gutleb, Arno C

    2016-02-17

    The increased incorporation of silver nanoparticles (Ag NPs) into consumer products makes the characterization of potential risk for humans and other organisms essential. The oral route is an important uptake route for NPs, therefore the study of the gastrointestinal tract in respect to NP uptake and toxicity is very timely. The aim of the present study was to evaluate the effects of Ag NPs and ions on a Caco-2/TC7:HT29-MTX intestinal co-culture model with mucus secretion, which constitutes an important protective barrier to exogenous agents in vivo and may strongly influence particle uptake. The presence of the mucus layer was confirmed with staining techniques (alcian blue and toluidine blue). Mono and co-cultures of Caco-2/TC7 and HT29-MTX cells were exposed to Ag NPs (Ag 20 and 200 nm) and AgNO3 and viability (alamar blue), ROS induction (DCFH-DA assay) and IL-8 release (ELISA) were measured. The particle agglomeration in the media was evaluated with DLS and the ion release with ultrafiltration and ICP-MS. The effects of the Ag NPs and AgNO3 on cells in co-culture were studied at a proteome level with two-dimensional difference in gel electrophoresis (2D-DIGE) followed by Matrix Assisted Laser Desorption Ionization - Time Of Flight/ Time Of Flight (MALDI-TOF/TOF) mass spectrometry (MS). Intracellular localization was assessed with NanoSIMS and TEM. The presence of mucus layer led to protection against ROS and decrease in IL-8 release. Both Ag 20 and 200 nm NPs were taken up by the cells and Ag NPs 20 nm were mainly localized in organelles with high sulfur content. A dose- and size-dependent increase in IL-8 release was observed with a lack of cytotoxicity and oxidative stress. Sixty one differentially abundant proteins were identified involved in cytoskeleton arrangement and cell cycle, oxidative stress, apoptosis, metabolism/detoxification and stress. The presence of mucus layer had an impact on modulating the induced toxicity of NPs. NP-specific effects were

  18. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    International Nuclear Information System (INIS)

    Suzuki, Hidenori; Taguchi, Toshihiko; Tanaka, Hiroshi; Kataoka, Hideo; Li Zhenglin; Muramatsu, Keiichi; Gondo, Toshikazu; Kawai, Shinya

    2004-01-01

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  19. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Science.gov (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  20. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Directory of Open Access Journals (Sweden)

    Sunita Nayak

    Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  1. Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model

    Energy Technology Data Exchange (ETDEWEB)

    Horie, Masafumi [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Saito, Akira, E-mail: asaitou-tky@umin.ac.jp [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Mikami, Yu [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan); Ohshima, Mitsuhiro [Department of Biochemistry, Ohu University School of Pharmaceutical Sciences (Japan); Morishita, Yasuyuki [Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo (Japan); Nakajima, Jun [Department of Thoracic Surgery, Graduate School of Medicine, University of Tokyo (Japan); Kohyama, Tadashi; Nagase, Takahide [Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We established three patient-paired sets of CAFs and NFs. Black-Right-Pointing-Pointer CAFs and NFs were analyzed using three-dimensional co-culture experiments. Black-Right-Pointing-Pointer CAFs clearly enhanced collagen gel contraction. Black-Right-Pointing-Pointer CAFs showed higher {alpha}-SMA expression than NFs. Black-Right-Pointing-Pointer CAFs were implicated in invasion and differentiation of lung cancer cells. -- Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher {alpha}-smooth muscle actin ({alpha}-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

  2. Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model

    International Nuclear Information System (INIS)

    Horie, Masafumi; Saito, Akira; Mikami, Yu; Ohshima, Mitsuhiro; Morishita, Yasuyuki; Nakajima, Jun; Kohyama, Tadashi; Nagase, Takahide

    2012-01-01

    Highlights: ► We established three patient-paired sets of CAFs and NFs. ► CAFs and NFs were analyzed using three-dimensional co-culture experiments. ► CAFs clearly enhanced collagen gel contraction. ► CAFs showed higher α-SMA expression than NFs. ► CAFs were implicated in invasion and differentiation of lung cancer cells. -- Abstract: Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

  3. In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone

    Directory of Open Access Journals (Sweden)

    Chiara Arrigoni

    2016-08-01

    Full Text Available Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps.

  4. Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system.

    Science.gov (United States)

    Kodama, Daisuke; Hirai, Takao; Kondo, Hisataka; Hamamura, Kazunori; Togari, Akifumi

    2017-02-01

    Recent studies have revealed that the sensory nervous system is involved in bone metabolism. However, the mechanism of communication between neurons and osteoblasts is yet to be elucidated. In this study, we investigated the signaling pathways between sensory neurons of the dorsal root ganglion (DRG) and the osteoblast-like MC3T3-E1 cells using an in vitro coculture system. Our findings indicate that signal transduction from DRG-derived neurons to MC3T3-E1 cells is suppressed by antagonists of the AMPA receptor and the NK 1 receptor. Conversely, signal transduction from MC3T3-E1 cells to DRG-derived neurons is suppressed by a P2X 7 receptor antagonist. Our results suggest that these cells communicate with each other by exocytosis of glutamate, substance P in the efferent signal, and ATP in the afferent signal. © 2017 Federation of European Biochemical Societies.

  5. Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage.

    Science.gov (United States)

    Dumont, Courtney M; Piselli, Jennifer M; Kazi, Nadeem; Bowman, Evan; Li, Guoyun; Linhardt, Robert J; Temple, Sally; Dai, Guohao; Thompson, Deanna M

    2017-08-15

    The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures. Endothelial factors generated under dynamic conditions significantly increased neuronal differentiation, while those released under static conditions stimulated oligodendrocyte differentiation. Flow increases EC release of neurogenic factors and of heparin sulfate glycosaminoglycans that increase their bioactivity, likely underlying the enhanced neuronal differentiation. Additionally, endothelial factors, especially from static conditions, promoted adherent growth. Together, our data suggest that blood flow may impact proliferation, adhesion, and the neuron-glial fate choice of adult NPCs, with implications for diseases and aging that reduce flow.

  6. Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS.

    Science.gov (United States)

    Kunze, Anja; Lengacher, Sylvain; Dirren, Elisabeth; Aebischer, Patrick; Magistretti, Pierre J; Renaud, Philippe

    2013-07-24

    Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

  7. Allelopathy in a leguminous mangrove plant, Derris indica: protoplast co-culture bioassay and rotenone effect.

    Science.gov (United States)

    Inoue, Aya; Mori, Daisuke; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2015-05-01

    To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

  8. Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

    Science.gov (United States)

    Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

    2015-02-01

    Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures.

    Science.gov (United States)

    Wu, Kun-Wei; Kou, Zeng-Wei; Mo, Jia-Lin; Deng, Xu-Xu; Sun, Feng-Yan

    2016-10-15

    This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (I A ) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (I K ) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced I A inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the I A . These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal I A by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Relative Abundances of Candida albicans and Candida glabrata in In Vitro Coculture Biofilms Impact Biofilm Structure and Formation.

    Science.gov (United States)

    Olson, Michelle L; Jayaraman, Arul; Kao, Katy C

    2018-04-15

    Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species- Candida albicans and Candida glabrata -are often coisolated from infection sites, suggesting the importance of Candida coculture biofilms. In this work, we report that biofilm formation of the coculture population depends on the relative ratio of starting cell concentrations of C. albicans and C. glabrata When using a starting ratio of C. albicans to C. glabrata of 1:3, ∼6.5- and ∼2.5-fold increases in biofilm biomass were observed relative to those of a C. albicans monoculture and a C. albicans / C. glabrata ratio of 1:1, respectively. Confocal microscopy analysis revealed the heterogeneity and complex structures composed of long C. albicans hyphae and C. glabrata cell clusters in the coculture biofilms, and reverse transcription-quantitative PCR (qRT-PCR) studies showed increases in the relative expression of the HWP1 and ALS3 adhesion genes in the C. albicans / C. glabrata 1:3 biofilm compared to that in the C. albicans monoculture biofilm. Additionally, only the 1:3 C. albicans / C. glabrata biofilm demonstrated an increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a coculture composition-dependent manner. IMPORTANCE Candida albicans and Candida glabrata are often coisolated during infection, and the occurrence of coisolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in

  11. Emergence of organized structure in co-culture spheroids: Experiments and Theory

    Science.gov (United States)

    Sanford, Roland; Kolbman, Dan; Song, Wei; Wu, Mingming; Ma, Minglin; Das, Moumita

    During tissue morphogenesis, from formation of embryos to tumor progression, cells often live and migrate in a heterogeneous environment consisting of many types of cells. To understand how differences in cell mechanobiological properties impact cellular self-organization and migration, we study a co-culture model composed of two distinct cell types confined in a three-dimensional spherical capsule. The cells are modeled as deformable, interacting, self-propelled particles that proliferate at specified timescales. A disordered potential is introduced to mimic the effect of the extracellular matrix (ECM). By varying the mechano-adhesive properties of each type, we investigate how differences in cell stiffness, cell-cell adhesion, and cell-ECM interaction influence collective properties of the binary cell population, such as self-assembly and migration. The predictions of the model are compared to experimental results on co-cutures of breast cancer cells and non-tumorigenic breast epithelial cells. This work was partially supported by a Cottrell College Science Award from the Research Corporation for Science Advancement.

  12. Endoglucanase and xylanase production by Bacillus sp. AR03 in co-culture.

    Science.gov (United States)

    Hero, Johan S; Pisa, José H; Perotti, Nora I; Romero, Cintia M; Martínez, María A

    2017-07-03

    The behavior of three isolates retrieved from different cellulolytic consortia, Bacillus sp. AR03, Paenibacillus sp. AR247 and Achromobacter sp. AR476-2, were examined individually and as co-cultures in order to evaluate their ability to produce extracellular cellulases and xylanases. Utilizing a peptone-based medium supplemented with carboxymethyl cellulose (CMC), an increase estimation of 1.30 and 1.50 times was obtained by the co-culture containing the strains AR03 and AR247, with respect to enzyme titles registered by their individual cultivation. On the contrary, the extracellular enzymatic production decreased during the co-cultivation of strain AR03 with the non-cellulolytic Achromobacter sp. AR476-2. The synergistic behavior observed through the combined cultivation of the strains AR03 and AR247 might be a consequence of the consumption by Paenibacillus sp. AR247 of the products of the CMC hydrolysis (i.e., cellobiose and/or cello-oligosaccharides), which were mostly generated by the cellulase producer Bacillus sp. AR03. The effect observed could be driven by the requirement to fulfill the nutritional supply from both strains on the substrate evaluated. These results would contribute to a better description of the degradation of the cellulose fraction of the plant cell walls in nature, expected to an efficient utilization of renewable sources.

  13. Substrate-limited co-culture for efficient production of propionic acid from flour hydrolysate.

    Science.gov (United States)

    Sabra, W; Dietz, D; Zeng, A P

    2013-07-01

    Propionic acid is presently mainly produced by chemical synthesis. For many applications, especially in feed and food industries, a fermentative production of propionic acid from cheap and renewable resources is of large interest. In this work, we investigated the use of a co-culture to convert household flour to propionic acid. Batch and fed-batch fermentations of hydrolyzed flour and a process of simultaneous saccharification and fermentation were examined and compared. Fed-batch culture with substrate limitation was found to be the most efficient process, reaching a propionic acid concentration of 30 g/L and a productivity of 0.33 g/L*h. This is the highest productivity so far achieved with free cells on media containing flour hydrolysate or glucose as carbon source. Batch culture and culture with controlled saccharification and fermentation delivered significantly lower propionic acid production (17-20 g/L) due to inhibition by the intermediate product lactate. It is concluded that co-culture fermentation of flour hydrolysate can be considered as an appealing bioprocess for the production of propionic acid.

  14. Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion

    Science.gov (United States)

    Happo, Mikko S.; Rönkkö, Teemu J.; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I.

    2018-01-01

    Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. PMID

  15. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts

    Science.gov (United States)

    Iskandar, Anita R.; Xiang, Yang; Frentzel, Stefan; Talikka, Marja; Leroy, Patrice; Kuehn, Diana; Guedj, Emmanuel; Martin, Florian; Mathis, Carole; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

    2015-01-01

    Organotypic 3D cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model. PMID:26085348

  16. The warburg effect in leukemia-stroma cocultures is mediated by mitochondrial uncoupling associated with uncoupling protein 2 activation.

    Science.gov (United States)

    Samudio, Ismael; Fiegl, Michael; McQueen, Teresa; Clise-Dwyer, Karen; Andreeff, Michael

    2008-07-01

    In 1956, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect (i.e., the inhibition of anaerobic fermentation by oxygen). Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here, we show that under normoxic conditions exposure of leukemia cells to bone marrow-derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (DeltaPsiM) in both cell types. Notably, the consumption of glucose was not altered in cocultures, suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in DeltaPsiM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas small interfering RNA-mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment activation of highly conserved mammalian UCPs may facilitate the Warburg effect in the absence of permanent respiratory impairment.

  17. Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

    Science.gov (United States)

    Lee, Hyeseung Janice

    Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and

  18. An ex vivo co-culture model system to evaluate stromal-epithelial interactions in breast cancer.

    Science.gov (United States)

    Salameh, T S; Le, T T; Nichols, M B; Bauer, E; Cheng, J; Camarillo, I G

    2013-01-15

    Breast cancer is the most commonly diagnosed cancer among women worldwide. High breast cancer incidence and mortality rates, especially in obese patients, emphasize the need for a better biological understanding of this disease. Previous studies provide substantial evidence for a vital role of the local extracellular environment in multiple steps of tumor progression, including proliferation and invasion. Current evidence supports the role of adipocytes as an endocrine organ, which produces steroid hormones, pro-inflammatory cytokines and adipokines, such as leptin. To further define the role of the mammary microenvironment on tumorigenesis, we have developed an adipose-tumor epithelial cell co-culture system designed to reproduce the in vivo mammary environment. We validate this model through use of coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. CARS analysis demonstrates the sustained viability of the adipocytes, and that mammary cancer cell morphology parallels that of tumors in vivo. Also, characterized was the influence of mammary adipose tissue on tumor cell growth and migration. Adipose tissue co-cultured with mammary tumor epithelial cells, in the absence of any serum or supplemental growth factors, resulted in substantial increases in growth and migration of tumor cells. In conclusion, this novel co-culture system provides an ideal model to study epithelial-stromal interactions in the mammary gland. Understanding the relationship between adipose tissue, the most abundant and least studied component of the breast stroma and tumor epithelial cells is critical to clarifying the influence of obesity on the development, progression and prognosis of breast cancer. Copyright © 2012 UICC.

  19. LowAgrobacterium tumefaciensinoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuusL.).

    Science.gov (United States)

    Zhang, Zhifen; Finer, John J

    2016-01-01

    Agrobacterium -mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 10 8 -10 9 bacteria mL -1 , followed by a 2-3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 10 2 bacteria mL -1 ) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 10 8 bacteria mL -1 ) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

  20. A 3D co-culture microtissue model of the human placenta for nanotoxicity assessment.

    Science.gov (United States)

    Muoth, Carina; Wichser, Adrian; Monopoli, Marco; Correia, Manuel; Ehrlich, Nicky; Loeschner, Katrin; Gallud, Audrey; Kucki, Melanie; Diener, Liliane; Manser, Pius; Jochum, Wolfram; Wick, Peter; Buerki-Thurnherr, Tina

    2016-10-06

    There is increasing evidence that certain nanoparticles (NPs) can overcome the placental barrier, raising concerns on potential adverse effects on the growing fetus. But even in the absence of placental transfer, NPs may pose a risk to proper fetal development if they interfere with the viability and functionality of the placental tissue. The effects of NPs on the human placenta are not well studied or understood, and predictive in vitro placenta models to achieve mechanistic insights on NP-placenta interactions are essentially lacking. Using the scaffold-free hanging drop technology, we developed a well-organized and highly reproducible 3D co-culture microtissue (MT) model consisting of a core of placental fibroblasts surrounded by a trophoblast cell layer, which resembles the structure of the in vivo placental tissue. We could show that secretion levels of human chorionic gonadotropin (hCG) were significantly higher in 3D than in 2D cell cultures, which indicates an enhanced differentiation of trophoblasts grown on 3D MTs. NP toxicity assessment revealed that cadmium telluride (CdTe) and copper oxide (CuO) NPs but not titanium dioxide (TiO 2 ) NPs decreased MT viability and reduced the release of hCG. NP acute toxicity was significantly reduced in 3D co-culture MTs compared to 2D monocultures. Taken together, 3D placental MTs provide a new and promising model for the fast generation of tissue-relevant acute NP toxicity data, which are indispensable for the safe development of NPs for industrial, commercial and medical applications.

  1. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics

    Directory of Open Access Journals (Sweden)

    Mansur Halai

    2014-09-01

    Full Text Available In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow–derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow–derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  2. Scanning electron microscopical observation of an osteoblast/osteoclast co-culture on micropatterned orthopaedic ceramics.

    Science.gov (United States)

    Halai, Mansur; Ker, Andrew; Meek, Rm Dominic; Nadeem, Danish; Sjostrom, Terje; Su, Bo; McNamara, Laura E; Dalby, Matthew J; Young, Peter S

    2014-01-01

    In biomaterial engineering, the surface of an implant can influence cell differentiation, adhesion and affinity towards the implant. On contact with an implant, bone marrow-derived mesenchymal stromal cells demonstrate differentiation towards bone forming osteoblasts, which can improve osteointegration. The process of micropatterning has been shown to improve osteointegration in polymers, but there are few reports surrounding ceramics. The purpose of this study was to establish a co-culture of bone marrow-derived mesenchymal stromal cells with osteoclast progenitor cells and to observe the response to micropatterned zirconia toughened alumina ceramics with 30 µm diameter pits. The aim was to establish whether the pits were specifically bioactive towards osteogenesis or were generally bioactive and would also stimulate osteoclastogenesis that could potentially lead to osteolysis. We demonstrate specific bioactivity of micropatterns towards osteogenesis, with more nodule formation and less osteoclastogenesis compared to planar controls. In addition, we found that that macrophage and osteoclast-like cells did not interact with the pits and formed fewer full-size osteoclast-like cells on the pitted surfaces. This may have a role when designing ceramic orthopaedic implants.

  3. A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-β1 treatment.

    Science.gov (United States)

    Ishikawa, Shinkichi; Ishimori, Kanae; Ito, Shigeaki

    2017-11-22

    The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated. We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) β1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis. TGF-β1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-β1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. Our findings indicate that TGF-β1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.

  4. Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture ...

    Indian Academy of Sciences (India)

    Prakash

    2009-12-09

    Dec 9, 2009 ... Colon carcinoma; HSP27; HSP72; MRP; IL-6; nitric oxide; co-culture. Expression of ... [Paduch R, Jakubowicz-Gil J and Kandefer-Szerszeń M 2009 Expression of HSP27, HSP72 and MRP proteins in in vitro co-culture of colon tumour ...... J, Jeannin J-F and Juillerat-Jeanneret L 2005 Evaluation of the.

  5. Staging Scenes of Co-Cultural Communication: Acting out Aspects of Marginalized and Dominant Identities

    Science.gov (United States)

    Root, Elizabeth

    2018-01-01

    Courses: Intercultural Communication, Interracial Communication, or an Interpersonal Communication class that covers co-cultural theory. Objectives: Students will be able to demonstrate a practical application of co-cultural theory by creating scenes that illustrate different communicative approaches and desired outcomes based on communication…

  6. The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes.

    Science.gov (United States)

    Seo, Seung-Beom; Choe, Eun Sang; Kim, Kwang-Sik; Shim, Soon-Mi

    2017-06-01

    Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood-brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC 50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4-16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.

  7. Pluripotency of adult stem cells derived from human and rat pancreas

    Science.gov (United States)

    Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

    Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

  8. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    Directory of Open Access Journals (Sweden)

    Tsutomu Motohashi

    2016-03-01

    Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

  9. Enhanced isopropanol and n-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate.

    Science.gov (United States)

    Zhang, Shaozhi; Qu, Chunyun; Huang, Xiaoyan; Suo, Yukai; Liao, Zhengping; Wang, Jufang

    2016-07-01

    The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.

  10. A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.

    Science.gov (United States)

    Le Berre-Scoul, Catherine; Chevalier, Julien; Oleynikova, Elena; Cossais, François; Talon, Sophie; Neunlist, Michel; Boudin, Hélène

    2017-01-15

    Unlike astrocytes in the brain, the potential role of enteric glial cells (EGCs) in the formation of the enteric neuronal circuit is currently unknown. To examine the role of EGCs in the formation of the neuronal network, we developed a novel neuron-enriched culture model from embryonic rat intestine grown in indirect coculture with EGCs. We found that EGCs shape axonal complexity and synapse density in enteric neurons, through purinergic- and glial cell line-derived neurotrophic factor-dependent pathways. Using a novel and valuable culture model to study enteric neuron-glia interactions, our study identified EGCs as a key cellular actor regulating neuronal network maturation. In the nervous system, the formation of neuronal circuitry results from a complex and coordinated action of intrinsic and extrinsic factors. In the CNS, extrinsic mediators derived from astrocytes have been shown to play a key role in neuronal maturation, including dendritic shaping, axon guidance and synaptogenesis. In the enteric nervous system (ENS), the potential role of enteric glial cells (EGCs) in the maturation of developing enteric neuronal circuit is currently unknown. A major obstacle in addressing this question is the difficulty in obtaining a valuable experimental model in which enteric neurons could be isolated and maintained without EGCs. We adapted a cell culture method previously developed for CNS neurons to establish a neuron-enriched primary culture from embryonic rat intestine which was cultured in indirect coculture with EGCs. We demonstrated that enteric neurons grown in such conditions showed several structural, phenotypic and functional hallmarks of proper development and maturation. However, when neurons were grown without EGCs, the complexity of the axonal arbour and the density of synapses were markedly reduced, suggesting that glial-derived factors contribute strongly to the formation of the neuronal circuitry. We found that these effects played by EGCs were

  11. Bactericidal Effect of Gold-Chitosan Nanocomposites in Coculture Models of Pathogenic Bacteria and Human Macrophages.

    Science.gov (United States)

    Mendoza, Gracia; Regiel-Futyra, Anna; Andreu, Vanesa; Sebastián, Víctor; Kyzioł, Agnieszka; Stochel, Grażyna; Arruebo, Manuel

    2017-05-31

    The ability of pathogenic bacteria to develop resistance mechanisms to avoid the antimicrobial potential of antibiotics has become an increasing problem for the healthcare system. The search for more effective and selective antimicrobial materials, though not harmful to mammalian cells, seems imperative. Herein we propose the use of gold-chitosan nanocomposites as effective bactericidal materials avoiding damage to human cells. Nanocomposites were obtained by taking advantage of the reductive and stabilizing action of chitosan solutions on two different gold precursor concentrations. The resulting nanocomposites were added at different final concentrations to a coculture model formed by Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria and human macrophages. Gold-chitosan colloids exhibited superior bactericidal ability against both bacterial models without showing cytotoxicity on human cells at the concentrations tested. Morphological and in vitro viability studies supported the feasibility of the infection model here described to test novel bactericidal nanomaterials. Flow cytometry and scanning electron microscopy analyses pointed to the disruption of the bacterial wall as the lethal mechanism. Data obtained in the present study suggest that gold-chitosan nanocomposites are powerful and promising nanomaterials for reducing bacteria-associated infections, respecting the integrity of mammalian cells, and displaying high selectivity against the studied bacteria.

  12. Interspecies electron transfer via hydrogen and formate rather than direct electrical connections in cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens

    DEFF Research Database (Denmark)

    Rotaru, Amelia-Elena; Shrestha, Pravin M; Liu, Fanghua

    2012-01-01

    was comparable in cocultures with that for the formate dehydrogenase mutant of G. sulfurreducens or the wild type, suggesting that H(2) was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains of G. sulfurreducens that could not produce pili or OmcS, two essential...

  13. Engineering E. coli-E. coli cocultures for production of muconic acid from glycerol.

    Science.gov (United States)

    Zhang, Haoran; Li, Zhengjun; Pereira, Brian; Stephanopoulos, Gregory

    2015-09-15

    cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of the E. coli-E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid. Two coculture engineering strategies were investigated. In the first strategy, an E. coli strain containing the complete biosynthesis pathway was co-cultivated with another E. coli strain containing only a heterologous intermediate-to-product biosynthetic pathway. In the second strategy, the upstream and downstream pathways were accommodated in two separate E. coli strains, each of which was dedicated to one portion of the biosynthesis process. Compared with the monoculture approach, both coculture engineering strategies improved the production significantly. Using a batch bioreactor, the engineered coculture achieved a 2 g/L muconic acid production with a yield of 0.1 g/g. Our results demonstrate that coculture engineering is a viable option for producing muconic acid from glycerol. Moreover, microbial coculture systems are shown to have the potential for converting single carbon source to value-added products.

  14. Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Shailesh S. Sawant

    2017-02-01

    Full Text Available Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs in pure cultures of marine bacterium, Saccharophagus degradans 2-40 (Sde 2-40, its contaminant, Bacillus cereus, and a co-culture of these bacteria (Sde 2-40 and B. cereus degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight. The present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.

  15. Comparative proteomic analysis of Methanothermobacter themautotrophicus ΔH in pure culture and in co-culture with a butyrate-oxidizing bacterium.

    Directory of Open Access Journals (Sweden)

    Miho Enoki

    Full Text Available To understand the physiological basis of methanogenic archaea living on interspecies H(2 transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H(2 supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F(420-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.

  16. Metabolomics Investigation of an Association of Induced Features and Corresponding Fungus during the Co-culture of Trametes versicolor and Ganoderma applanatum

    Science.gov (United States)

    Xu, Xiao-Yan; Shen, Xiao-Ting; Yuan, Xiao-Jie; Zhou, Yuan-Ming; Fan, Huan; Zhu, Li-Ping; Du, Feng-Yu; Sadilek, Martin; Yang, Jie; Qiao, Bin; Yang, Song

    2018-01-01

    The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current work sets

  17. Metabolomics Investigation of an Association of Induced Features and Corresponding Fungus during the Co-culture of Trametes versicolor and Ganoderma applanatum

    Directory of Open Access Journals (Sweden)

    Xiao-Yan Xu

    2018-01-01

    Full Text Available The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor, 6 from G. applanatum, and 5 features were synthesized by both T. versicolor and G. applanatum. 13C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenylformamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13C incorporation in the mono-culture of both T. versicolor and G. applanatum, was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The

  18. Metabolomics Investigation of an Association of Induced Features and Corresponding Fungus during the Co-culture of Trametes versicolor and Ganoderma applanatum.

    Science.gov (United States)

    Xu, Xiao-Yan; Shen, Xiao-Ting; Yuan, Xiao-Jie; Zhou, Yuan-Ming; Fan, Huan; Zhu, Li-Ping; Du, Feng-Yu; Sadilek, Martin; Yang, Jie; Qiao, Bin; Yang, Song

    2017-01-01

    The co-culture of Trametes versicolor and Ganoderma applanatum is a model of intense basidiomycete interaction, which induces many newly synthesized or highly produced features. Currently, one of the major challenges is an identification of the origin of induced features during the co-culture. Herein, we report a 13 C-dynamic labeling analysis used to determine an association of induced features and corresponding fungus even if the identities of metabolites were not available or almost nothing was known of biochemical aspects. After the co-culture of T. versicolor and G. applanatum for 10 days, the mycelium pellets of T. versicolor and G. applanatum were sterilely harvested and then mono-cultured in the liquid medium containing half fresh medium with 13 C-labeled glucose as carbon source and half co-cultured supernatants collected on day 10. 13 C-labeled metabolome analyzed by LC-MS revealed that 31 induced features including 3-phenyllactic acid and orsellinic acid were isotopically labeled in the mono-culture after the co-culture stimulation. Twenty features were derived from T. versicolor , 6 from G. applanatum , and 5 features were synthesized by both T. versicolor and G. applanatum . 13 C-labeling further suggested that 12 features such as previously identified novel xyloside [N-(4-methoxyphenyl)formamide 2-O-beta-D-xyloside] were likely induced through the direct physical interaction of mycelia. Use of molecular network analysis combined with 13 C-labeling provided an insight into the link between the generation of structural analogs and producing fungus. Compound 1 with m/z 309.0757, increased 15.4-fold in the co-culture and observed 13 C incorporation in the mono-culture of both T. versicolor and G. applanatum , was purified and identified as a phenyl polyketide, 2,5,6-trihydroxy-4, 6-diphenylcyclohex-4-ene-1,3-dione. The biological activity study indicated that this compound has a potential to inhibit cell viability of leukemic cell line U937. The current

  19. A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kostadinova, Radina; Boess, Franziska [Non-Clinical Safety, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, Building 73 / Room 117b, 4070 Basel (Switzerland); Applegate, Dawn [RegeneMed, 9855 Towne Centre Drive Suite 200, San Diego, CA 92121 (United States); Suter, Laura; Weiser, Thomas; Singer, Thomas [Non-Clinical Safety, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, Building 73 / Room 117b, 4070 Basel (Switzerland); Naughton, Brian [RegeneMed, 9855 Towne Centre Drive Suite 200, San Diego, CA 92121 (United States); Roth, Adrian, E-mail: adrian_b.roth@roche.com [Non-Clinical Safety, Hoffmann-La Roche Ltd, Grenzacherstrasse 124, Building 73 / Room 117b, 4070 Basel (Switzerland)

    2013-04-01

    Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3 months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures. In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects. - Highlights: ► 3D liver co-cultures maintain liver specific functions for up to three months. ► Activities of Cytochrome P450s remain drug- inducible accross three months. ► 3D liver co-cultures recapitulate drug-induced liver toxicity

  20. A long-term three dimensional liver co-culture system for improved prediction of clinically relevant drug-induced hepatotoxicity

    International Nuclear Information System (INIS)

    Kostadinova, Radina; Boess, Franziska; Applegate, Dawn; Suter, Laura; Weiser, Thomas; Singer, Thomas; Naughton, Brian; Roth, Adrian

    2013-01-01

    Drug-induced liver injury (DILI) is the major cause for liver failure and post-marketing drug withdrawals. Due to species-specific differences in hepatocellular function, animal experiments to assess potential liabilities of drug candidates can predict hepatotoxicity in humans only to a certain extent. In addition to animal experimentation, primary hepatocytes from rat or human are widely used for pre-clinical safety assessment. However, as many toxic responses in vivo are mediated by a complex interplay among different cell types and often require chronic drug exposures, the predictive performance of hepatocytes is very limited. Here, we established and characterized human and rat in vitro three-dimensional (3D) liver co-culture systems containing primary parenchymal and non-parenchymal hepatic cells. Our data demonstrate that cells cultured on a 3D scaffold have a preserved composition of hepatocytes, stellate, Kupffer and endothelial cells and maintain liver function for up to 3 months, as measured by the production of albumin, fibrinogen, transferrin and urea. Additionally, 3D liver co-cultures maintain cytochrome P450 inducibility, form bile canaliculi-like structures and respond to inflammatory stimuli. Upon incubation with selected hepatotoxicants including drugs which have been shown to induce idiosyncratic toxicity, we demonstrated that this model better detected in vivo drug-induced toxicity, including species-specific drug effects, when compared to monolayer hepatocyte cultures. In conclusion, our results underline the importance of more complex and long lasting in vitro cell culture models that contain all liver cell types and allow repeated drug-treatments for detection of in vivo-relevant adverse drug effects. - Highlights: ► 3D liver co-cultures maintain liver specific functions for up to three months. ► Activities of Cytochrome P450s remain drug- inducible accross three months. ► 3D liver co-cultures recapitulate drug-induced liver toxicity

  1. Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks.

    Science.gov (United States)

    Aebersold, Mathias J; Thompson-Steckel, Greta; Joutang, Adriane; Schneider, Moritz; Burchert, Conrad; Forró, Csaba; Weydert, Serge; Han, Hana; Vörös, János

    2018-01-01

    Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm 2 down to isolated cells at 1,000 cells/cm 2 . Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development.

  2. Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

    Directory of Open Access Journals (Sweden)

    Mathias J. Aebersold

    2018-02-01

    Full Text Available Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development.

  3. Behaviour of Kloeckera apiculata Flocculent Strain in Coculture with Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Oscar A. Sosa

    2008-01-01

    Full Text Available The behaviour of the flocculent Kloeckera apiculata strain in coculture with the nonflocculent Saccharomyces cerevisiae strain, both yeasts isolated from wine, has been analyzed. Pure culture of Kloeckera apiculata (apiculate yeast exhibits 75 % flocculation in the yeast extract/peptone/glucose (containing 20 g/L of glucose, YPG20 medium after 24-hour incubation at 28 °C. This yeast expresses a weak flocculent phenotype in glucose-poor medium (10 % of flocculated cells. Increasing the glucose concentrations in this medium induces yeast flocculation. When the apiculate yeast was co-inoculated with a nonflocculent strain of Saccharomyces cerevisiae (elliptic yeast, an increase of the number of elliptic cells that settled at the bottom of the culture was observed. Electron microscopy observations of the aggregates formed in the mixed culture confirmed that apiculate and elliptic yeasts can interact and establish a binding between them through homogeneous mucus. However, a lower percentage of Kloeckera apiculata flocculation with respect to the pure cultures was observed, this behaviour being correlated with a higher rate of glucose consumption by Saccharomyces cerevisiae. The prompt coflocculation of Kloeckera apiculata with Saccharomyces strains and the induction of this phenomenon by glucose could be considered an important biotechnological tool for the early decreasing of indigenous saccharomycetic flora from the media, before the inoculation of a selected starter strain to carry out a more controlled alcohol fermentation.

  4. Where the thoughts dwell: The physiology of neuronal-glial "diffuse neural net"

    Czech Academy of Sciences Publication Activity Database

    Verkhratsky, Alexei; Parpura, V.; Rodríguez Arellano, Jose Julio

    2011-01-01

    Roč. 66, 1-2 (2011), s. 133-151 ISSN 0165-0173 R&D Projects: GA ČR GA309/09/1696; GA ČR GA305/08/1384 Institutional research plan: CEZ:AV0Z50390703 Keywords : human brain * glia * neurone Subject RIV: FH - Neurology Impact factor: 10.342, year: 2011

  5. Neuronismo y reticulismo: Neuronal-glial circuits unify the reticular and neuronal theories of brain organisation

    Czech Academy of Sciences Publication Activity Database

    Verkhratsky, Alexei

    2009-01-01

    Roč. 195, č. 1 (2009), s. 111-122 ISSN 1748-1708 Institutional research plan: CEZ:AV0Z50390512 Keywords : gliotransmitters * neurotransmitters * history of neuroscience Subject RIV: FH - Neurology Impact factor: 2.810, year: 2009

  6. Production of rhamnolipids by Pseudomonas aeruginosa is inhibited by H2S but resumes in a co-culture with P. stutzeri: applications for microbial enhanced oil recovery.

    Science.gov (United States)

    Zhao, Feng; Ma, Fang; Shi, Rongjiu; Zhang, Jie; Han, Siqin; Zhang, Ying

    2015-09-01

    Sulfate-reducing bacteria and H2S exist widely in oil production systems, and in situ production of rhamnolipids is promising for microbial enhanced oil recovery (MEOR). However, information of the effect of S(2-) on rhamnolipids production is scarce. Two facultative anaerobic rhamnolipids-producing bacterial strains, Pseudomonas aeruginosa SG and WJ-1, were used. Above 10 mg S(2-)/l, both cell growth and rhamnolipids production were inhibited. A large inoculum (9%, v/v) failed to completely relieve the inhibitory effect of 10 mg S(2-)/l. Below 30 mg S(2-)/l, both strains resumed rhamnolipid production through co-culturing with the denitrifying and sulphide-removing strain Pseudomonas stutzeri DQ1. H2S has a direct but reversible inhibitory effect on rhamnolipids production. Control of H2S in oilfields is indispensable to MEOR, and the co-culture method is effective in restoring rhamnolipid production in presence of S(2-).

  7. Optimized simultaneous saccharification and co-fermentation of rice straw for ethanol production by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture using design of experiments.

    Science.gov (United States)

    Suriyachai, Nopparat; Weerasaia, Khatiya; Laosiripojana, Navadol; Champreda, Verawat; Unrean, Pornkamol

    2013-08-01

    Herein an ethanol production process from rice straw was optimized. Simultaneous saccharification and co-fermentation (SSCF) using Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture was carried out to enhance ethanol production. The optimal saccharification solid loading was 5%. Key fermentation parameters for co-culture including cell ratio, agitation rate and temperature was rationally optimized using design of experiment (DoE). Optimized co-culture conditions for maximum ethanol production efficiency were at S. cerevisiae:S. stipitis cell ratio of 0.31, agitation rate of 116 rpm and temperature of 33.1°C. The optimized SSCF process reached ethanol titer of 15.2g/L and ethanol yield of 99% of theoretical yield, consistent with the DoE model prediction. Moreover, SSCF process under high biomass concentration resulted in high ethanol concentration of 28.6g/L. This work suggests the efficiency and scalability of the developed SSCF process which could provide an important basis for the economic feasibility of ethanol production from lignocelluloses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Bioethanol production from sweet potato (Ipomoea batatas L. flour using co-culture of Trichoderma sp. and Saccharomyces cerevisiae in solid-state fermentation

    Directory of Open Access Journals (Sweden)

    Manas Ranjan Swain

    2013-04-01

    Full Text Available The aim of this work was to study the optimiation of co-culturing of Trichoderma sp. and Saccharomyces cerevisiae (1:4 ratio on sweet potato (Ipomoea batatas L. flour (SPF for the production of bio-ethanol in solid-state fermentation (SSF. Maximum ethanol (172 g/kg substrate was produced in a medium containing 80% moisture, ammonium sulphate 0.2%, pH 5.0, inoculuted with 10% inoculum size and fermented at 30ºC for 72h. .Concomitant with highest ethanol concentration, maximum ethanol productivity (2.8 g/kg substrate/h, microbial biomass (23×10(8 CFU/ g substrate, ethanol yield (47 g/100g sugar consumed and fermentation efficiency (72% were also obtained under these conditions. Cell interaction was observed familiar between the viable cells of Trichoderma sp. and S. cerevisiae when co-cultured. Ethanol production ability by the co-culture was 65 % higher than the single culture of S. cerevisiae from un-saccharified SPF.

  9. Production of butanol by fermentation in the presence of cocultures of clostridium

    Science.gov (United States)

    Bergstrom, S. L.; Foutch, G. L. (Inventor)

    1985-01-01

    Sugars are converted to a mixture of solvents including butanol by a fermentation process employing a coculture of microorganisms of the Clostridium genus, one of said microorganisms favoring the production of butyric acid and the other of which converts the butyric acid so produced to butanol. The use of a coculture substantially increases the yield of butanol over that obtained using a culture employing only one microorganism.

  10. Decolorization of synthetic brilliant green carpet industry dye through fungal co-culture technology.

    Science.gov (United States)

    Kumari, Simpal; Naraian, Ram

    2016-09-15

    Aim of the present study was to evaluate the efficiency of fungal co-culture for the decolorization of synthetic brilliant green carpet industry dye. For this purpose two lignocellulolytic fungi Pleurotus florida (PF) and Rhizoctonia solani (RS) were employed. The study includes determination of enzyme profiles (laccase and peroxidase), dye decolorization efficiency of co-culture and crude enzyme extracts. Both fungi produced laccase and Mn peroxidase and successfully decolorized solutions of different concentrations (2.0, 4.0, 6.0, & 8.0(w/v) of dye. The co-culture resulted highest 98.54% dye decolorization at 2% (w/v) of dye as compared to monocultures (82.12% with PF and 68.89% with RS) during 12 days of submerged fermentation. The lower levels of dyes were rapidly decolorized, while higher levels in slow order as 87.67% decolorization of 8% dye. The promising achievement of the study was remarkable decolorizing efficiency of co-culture over monocultures. The direct treatment of the mono and co-culture enzyme extracts to dye also influenced remarkable. The highest enzymatic decolorization was through combined (PF and RS) extracts, while lesser by monoculture extracts. Based on the observations and potentiality of co-culture technology; further it can be exploited for the bioremediation of areas contaminated with hazardous environmental pollutants including textile and other industry effluents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact.

    Science.gov (United States)

    Takigawa, Hidehiko; Kitadai, Yasuhiko; Shinagawa, Kei; Yuge, Ryo; Higashi, Yukihito; Tanaka, Shinji; Yasui, Wataru; Chayama, Kazuaki

    2017-05-01

    We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow-derived mesenchymal stem cells (MSCs) migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT)-related genes such as fibronectin (FN), SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Axon ensheathment and metabolic supply by glial cells in Drosophila.

    Science.gov (United States)

    Schirmeier, Stefanie; Matzat, Till; Klämbt, Christian

    2016-06-15

    Neuronal function requires constant working conditions and a well-balanced supply of ions and metabolites. The metabolic homeostasis in the nervous system crucially depends on the presence of glial cells, which nurture and isolate neuronal cells. Here we review recent findings on how these tasks are performed by glial cells in the genetically amenable model organism Drosophila melanogaster. Despite the small size of its nervous system, which would allow diffusion of metabolites, a surprising division of labor between glial cells and neurons is evident. Glial cells are glycolytically active and transfer lactate and alanine to neurons. Neurons in turn do not require glycolysis but can use the glially provided compounds for their energy homeostasis. Besides feeding neurons, glial cells also insulate neuronal axons in a way similar to Remak fibers in the mammalian nervous system. The molecular mechanisms orchestrating this insulation require neuregulin signaling and resemble the mechanisms controlling glial differentiation in mammals surprisingly well. We hypothesize that metabolic cross talk and insulation of neurons by glial cells emerged early during evolution as two closely interlinked features in the nervous system. This article is part of a Special Issue entitled SI: Myelin Evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Extracellular Membrane Vesicles as Vehicles for Brain Cell-to-Cell Interactions in Physiological as well as Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Gabriella Schiera

    2015-01-01

    Full Text Available Extracellular vesicles are involved in a great variety of physiological events occurring in the nervous system, such as cross talk among neurons and glial cells in synapse development and function, integrated neuronal plasticity, neuronal-glial metabolic exchanges, and synthesis and dynamic renewal of myelin. Many of these EV-mediated processes depend on the exchange of proteins, mRNAs, and noncoding RNAs, including miRNAs, which occurs among glial and neuronal cells. In addition, production and exchange of EVs can be modified under pathological conditions, such as brain cancer and neurodegeneration. Like other cancer cells, brain tumours can use EVs to secrete factors, which allow escaping from immune surveillance, and to transfer molecules into the surrounding cells, thus transforming their phenotype. Moreover, EVs can function as a way to discard material dangerous to cancer cells, such as differentiation-inducing proteins, and even drugs. Intriguingly, EVs seem to be also involved in spreading through the brain of aggregated proteins, such as prions and aggregated tau protein. Finally, EVs can carry useful biomarkers for the early diagnosis of diseases. Herein we summarize possible roles of EVs in brain physiological functions and discuss their involvement in the horizontal spreading, from cell to cell, of both cancer and neurodegenerative pathologies.

  14. Functional genomic study of the environmentally important Desulfovibrio /Methanococcus syntrophic co-culture.

    Science.gov (United States)

    Mukhopadhyay, A.

    2008-12-01

    The use of microbe-oriented bioremediation for ameliorating extensive environmental pollution has fostered fundamental and applied studies of environmentally relevant microorganisms such as Desulfovibrio vulgaris, Shewanella oneidensis and Geobacter metallireducens.. Concurrently, there has been an increasing appreciation that the physiology of these organisms in pure culture is not necessarily representative of its activities in the environment. To enable a better understanding of microbial physiology under more environmentally relevant conditions, the syntrophic growth between the sulfate reducing bacterium, D. vulgaris and the hydrogenotrophic methanogen, Methanococcus maripaludis serves as an ideal system for laboratory studies. Cell wide analyses using transcript, proteomics and metabolite analysis have been widely used to understand cellular activity at a molecular level. Using D. vulgaris and M. maripaludis arrays, and the iTRAQ proteomics method, we studied the physiology of the D. vulgaris / M. maripaludis syntrophic co- cultures. The results from this study allowed us to identify differences in cellular response in mono-culture vs. co-culture growth for both D. vulgaris and M. maripaludis.

  15. Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

    Directory of Open Access Journals (Sweden)

    Tatiana Takahasi Komoto

    2015-01-01

    Full Text Available Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of T. rubrum cocultured with keratinocytes and treated with the flavonoid trans-chalcone and the glycoalkaloid α-solanine. Both substances showed a marked antifungal activity against T. rubrum strain CBS (MIC = 1.15 and 17.8 µg/mL, resp.. Cytotoxicity assay against HaCaT cells produced IC50 values of 44.18 to trans-chalcone and 61.60 µM to α-solanine. The interaction of keratinocytes with T. rubrum conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter TruMDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and TruMDR2 genes. Furthermore, the trans-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.

  16. The Dietary Isoflavone Daidzein Reduces Expression of Pro-Inflammatory Genes through PPARα/γ and JNK Pathways in Adipocyte and Macrophage Co-Cultures.

    Directory of Open Access Journals (Sweden)

    Yuri Sakamoto

    Full Text Available Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance, and peroxisome proliferator-activated receptors (PPARs regulate inflammatory gene expression in these cells. Recently, the soy isoflavone daidzein was reported to act as a PPAR activator. We examined whether daidzein affected adipocyte-macrophage crosstalk via the regulation of PPARs. Co-cultures of 3T3-L1 adipocytes and RAW264 macrophages, or palmitate-stimulated RAW264 macrophages were treated with daidzein in the presence or absence of specific inhibitors for PPARs: GW6471 (a PPARα antagonist, and GW9662 (a PPARγ antagonist. Inflammatory gene expression was then determined. Daidzein significantly decreased chemokine (C-C motif ligand 2 (Ccl2, known in humans as monocyte chemo-attractant protein 1 (MCP1 and interleukin 6 (Il6 mRNA levels induced by co-culture. In 3T3-L1 adipocytes, daidzein inversed the attenuation of adiponectin gene expression by co-culture, and these effects were inhibited by the PPAR-γ specific inhibitor. Daidzein also decreased Ccl2 and Il6 mRNA levels in RAW264 macrophages stimulated with palmitate or conditioned medium (CM from hypertrophied 3T3-L1 adipocytes. This inhibitory effect on Il6 expression was abrogated by a PPAR-α inhibitor. Additionally, we examined the activation of nuclear factor-kappa B (NF-κB and c-Jun N-terminal kinase (JNK pathways and found that daidzein significantly inhibited palmitate-induced phosphorylation of JNK. Our data suggest that daidzein regulates pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the JNK pathway in adipocyte and macrophage co-cultures. These effects might be favorable in improving adipose inflammation, thus, treatment of daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue.

  17. Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

    Science.gov (United States)

    Al-Shehri, Abdulghani; Favretto, Marco E; Ioannou, Panayiotis V; Romero, Ignacio A; Couraud, Pierre-Olivier; Weksler, Babette Barbash; Parker, Terry L; Kallinteri, Paraskevi

    2015-03-01

    Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

  18. SOCS3 in retinal neurons and glial cells suppresses VEGF signaling to prevent pathological neovascular growth.

    Science.gov (United States)

    Sun, Ye; Ju, Meihua; Lin, Zhiqiang; Fredrick, Thomas W; Evans, Lucy P; Tian, Katherine T; Saba, Nicholas J; Morss, Peyton C; Pu, William T; Chen, Jing; Stahl, Andreas; Joyal, Jean-Sébastien; Smith, Lois E H

    2015-09-22

    Neurons and glial cells in the retina contribute to neovascularization, or the formation of abnormal new blood vessels, in proliferative retinopathy, a condition that can lead to vision loss or blindness. We identified a mechanism by which suppressor of cytokine signaling 3 (SOCS3) in neurons and glial cells prevents neovascularization. We found that Socs3 expression was increased in the retinal ganglion cell and inner nuclear layers after oxygen-induced retinopathy. Mice with Socs3 deficiency in neuronal and glial cells had substantially reduced vaso-obliterated retinal areas and increased pathological retinal neovascularization in response to oxygen-induced retinopathy, suggesting that loss of neuronal/glial SOCS3 increased both retinal vascular regrowth and pathological neovascularization. Furthermore, retinal expression of Vegfa (which encodes vascular endothelial growth factor A) was higher in these mice than in Socs3 flox/flox controls, indicating that neuronal and glial SOCS3 suppressed Vegfa expression during pathological conditions. Lack of neuronal and glial SOCS3 resulted in greater phosphorylation and activation of STAT3, which led to increased expression of its gene target Vegfa, and increased endothelial cell proliferation. In summary, SOCS3 in neurons and glial cells inhibited the STAT3-mediated secretion of VEGF from these cells, which suppresses endothelial cell activation, resulting in decreased endothelial cell proliferation and angiogenesis. These results suggest that neuronal and glial cell SOCS3 limits pathological retinal angiogenesis by suppressing VEGF signaling. Copyright © 2015, American Association for the Advancement of Science.

  19. Roles of glial cells in schizophrenia: possible targets for therapeutic approaches.

    Science.gov (United States)

    Takahashi, Nagahide; Sakurai, Takeshi

    2013-05-01

    Glial cells consisting of oligodendrocytes, astrocytes, microglia, and NG2 positive cells are major cell populations in the central nervous system, number-wise. They function as effectors and modulators of neurodevelopment through a wide variety of neuron-glial cell interactions in brain development and functions. Glial cells can be affected by both genetic and environmental factors, leading to their dysfunctions in supporting neuronal development and functions. These in turn can affect neuronal cells, causing alterations at the circuitry level that manifest as behavioral characteristics associated with schizophrenia in late teens-early twenties. Glial cells are also involved in neuroinflammatory processes, which sometimes have deleterious effects on the normal brain development. If the glial involvement plays significant roles in schizophrenia, the processes involving glial cells can become possible therapeutic targets for schizophrenia. A number of known antipsychotics are shown to have beneficial effects on glial cells, but other drugs targeting glial cell functions may also have therapeutic effects on schizophrenia. The latter can be taken into consideration for future drug development for schizophrenia. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Functional investigation of NCI-H460-inducible myofibroblasts on the chemoresistance to VP-16 with a microfluidic 3D co-culture device.

    Science.gov (United States)

    Hao, Yuanyuan; Zhang, Lichuan; He, Jiarui; Guo, Zhe; Ying, Li; Xu, Zhiyun; Zhang, Jianing; Lu, Jianxin; Wang, Qi

    2013-01-01

    Fibroblasts, the major cell type in tumor stroma, are essential for tumor growth and survival, and represent an important therapeutic target for cancers. Here we presented a microfluidic co-culture device in which the three-dimensional (3D) matrix was employed to reconstruct an in vivo-like fibroblast-tumor cell microenvironment for investigation of the role of myofibroblasts induced by lung cancer cells in the chemoresistance to VP-16. Composed of a double-layer chip and an injection pump, the device houses fibroblasts and lung cancer cells co-cultured in 3D matrix and 2D mode to induce fibroblasts to become myofibroblasts with the supplement of the medium continuously. With this device, we verified that the cytokines secreted by lung cancer cells could effectively transform the fibroblasts into myofibroblasts. Moreover, compared to fibroblasts, the myofibroblasts showed higher resistance to anticancer drug VP-16. We also demonstrated that this kind of acquired resistance in myofibroblasts was associated with the expression of Glucose-regulated protein 78 (GP78). We concluded that this device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.

  1. Garlic exerts allelopathic effects on pepper physiology in a hydroponic co-culture system

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    Haiyan Ding

    2016-05-01

    Full Text Available A hydroponic co-culture system was adopted to determine the allelopathic potential of garlic on the growth of pepper plants. Different numbers of garlic plants (0, 2, 4, 8 and 12 were hydroponically co-cultured with two pepper plants to investigate allelopathic effects on the growth attributes and antioxidative defense system of the test pepper plants. The responses of the pepper plants depended on the number of garlic plants included in the co-culture system, indicating an association of pepper growth with the garlic root exudate concentration. When grown at a pepper/garlic ratio of 1:1 or 1:2, the pepper plant height, chlorophyll content, and peroxidase (POD, catalase (CAT and phenylalanine ammonia-lyase (PAL activities were significantly increased after 30 days of co-culture; in contrast, reduction in methane dicarboxylic aldehyde (MDA content was observed. However, when the pepper/garlic ratio was 1:4 or higher, these morphological indices and protective enzyme activities were significantly inhibited, whereas MDA levels in the pepper leaves were significantly increased due to severe membrane lipid peroxidation. The results indicate that although low concentrations of garlic root exudates appear to induce protective enzyme systems and promote pepper growth, high concentrations have deleterious effects. These findings suggest that further investigations should optimize the co-culture pepper/garlic ratio to reduce continuous cropping obstacles in pepper production.

  2. High-temperature ethanol fermentation by immobilized coculture of Kluyveromyces marxianus and Saccharomyces cerevisiae.

    Science.gov (United States)

    Eiadpum, Akekasit; Limtong, Savitree; Phisalaphong, Muenduen

    2012-09-01

    Suspended and immobilized cocultures of the thermotolerant yeast, Kluyveromyces marxianus DMKU 3-1042 and the mesophilic flocculent yeast, Saccharomyces cerevisiae M30 were studied for their abilities to improve production and stability of ethanol fermentation. Sugarcane juice and blackstrap molasses, at initial sugar concentrations of 220 g/L, were used as carbon sources. The results indicated that the coculture system could improve ethanol production from both sugarcane juice and blackstrap molasses when the operating temperature ranged between 33 °C and 45 °C. High temperature tolerances were achieved when the coculture was immobilized. The immobilized coculture was more effective in high-temperature ethanol fermentation than the suspended cultures. The coculture immobilized on thin-shell silk cocoon and fermented at 37 °C and 40 °C generated maximal ethanol concentrations of 81.4 and 77.3 g/L, respectively, which were 5.9-8.7% and 16.8-39.0% higher than those of the suspended cultures, respectively. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

    Directory of Open Access Journals (Sweden)

    Ertl Ronald F

    2001-09-01

    Full Text Available Abstract Background Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels. Methods Blood monocytes from healthy donors and human fetal lung fibroblasts were cast into type I collagen gels and maintained in floating cultures for three weeks. Results Fibroblast-mediated gel contraction was initially inhibited by the presence of monocytes (P P P 2 production was significantly increased by co-culture and its presence attenuated collagen degradation. Conclusion The current study, therefore, demonstrates that interaction between monocytes and fibroblasts can contract and degrade extracellular matrix in extended culture.

  4. Differential expression of stem cell-like proteins in normal, hyperplastic and dysplastic oral epithelium

    Directory of Open Access Journals (Sweden)

    Sarah Mohammed Mohammed BARAKAT

    2015-02-01

    Full Text Available Objective The identification of stem cells (SC remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche, but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia. Material and Methods Thirty oral epithelial dysplasia (OED, 25 oral lichen planus (OLP, 10 oral hyperkeratosis and 5 normal oral epithelium (OE were immunohistochemically examined for four SC markers [integrin β1, neuron-glial-2 (NG2, notch 1 (N1 and keratin 15 (K15]. Results Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin β1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker. Conclusions Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further.

  5. Differential expression of stem cell-like proteins in normal, hyperplastic and dysplastic oral epithelium.

    Science.gov (United States)

    Barakat, Sarah Mohammed Mohammed; Siar, Chong Huat

    2015-01-01

    The identification of stem cells (SC) remains challenging. In the human oral mucosal epithelium, these cells are believed to be in the basal layer (stem cell niche), but their exact location is unclear. The aim of this study was to examine the dysplastic oral epithelium for these SC-like proteins in order to assess their diagnostic value as biomarkers complementing the histological grading of dysplasia. Thirty oral epithelial dysplasia (OED), 25 oral lichen planus (OLP), 10 oral hyperkeratosis and 5 normal oral epithelium (OE) were immunohistochemically examined for four SC markers [integrin β1, neuron-glial-2 (NG2), notch 1 (N1) and keratin 15 (K15)]. Three of four SC markers were heterogeneously detected in all samples. K15 overexpression in the lower two-thirds of severe OED suggests an expanded SC niche. Integrin β1 distribution pattern was not measurably different between OEDs and control. NG2 was almost negative to absent in all samples examined. N1 expression was weak and highly variable in normal and dysplastic epithelium, making it an unreliable epithelial stem cell marker. Present findings suggest that these markers were unable to identify individual epithelial stem cells. Instead, subpopulations of cells, most probably stem cells and transit amplifying cells with stem cell-like properties were identified in the dysplastic oral epithelium. The characteristic expressions of K15 might be of diagnostic value for oral dysplasia and should be investigated further.

  6. Cryopreservation of Hair-Follicle Associated Pluripotent (HAP) Stem Cells Maintains Differentiation and Hair-Growth Potential.

    Science.gov (United States)

    Hoffman, Robert M; Kajiura, Satoshi; Cao, Wenluo; Liu, Fang; Amoh, Yasuyuki

    2016-01-01

    Hair follicles contain nestin-expressing pluripotent stem cells which originate above the bulge area of the follicle, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. We have established efficient cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells as well as hair growth. We cryopreserved the whole hair follicle by slow-rate cooling in TC-Protector medium or in DMSO-containing medium and storage in liquid nitrogen or at -80 °C. After thawing and culture of the cryopreserved whisker follicles, growing HAP stem cells formed hair spheres. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. We have also previously demonstrated that cryopreserved mouse whisker hair follicles maintain their hair-growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. DMSO-cryopreserved hair follicles also maintained the HAP stem cells, evidenced by P75 ntr expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair-shaft growth of cryopreserved hair follicles. HAP stem cells can be used for nerve and spinal-cord repair. This biobanking of hair follicles can allow each patient the potential for their own stem cell use for regenerative medicine or hair transplantation.

  7. Microbial consortia: a critical look at microalgae co-cultures for enhanced biomanufacturing.

    Science.gov (United States)

    Padmaperuma, Gloria; Kapoore, Rahul Vijay; Gilmour, Daniel James; Vaidyanathan, Seetharaman

    2017-12-12

    Monocultures have been the preferred production route in the bio-industry, where contamination has been a major bottleneck. In nature, microorganisms usually exist as part of organized communities and consortia, gaining benefits from co-habitation, keeping invaders at bay. There is increasing interest in the use of co-cultures to tackle contamination issues, and simultaneously increase productivity and product diversity. The feasibility of extending the natural phenomenon of co-habitation to the biomanufacturing industry in the form of co-cultures requires careful and systematic consideration of several aspects. This article will critically examine and review current work on microbial co-cultures, with the intent of examining the concept and proposing a design pipeline that can be developed in a biomanufacturing context.

  8. Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Alicia M. Barnett

    2016-05-01

    Full Text Available Caprine milk contains the highest amount of oligosaccharides among domestic animals, which are structurally similar to human milk oligosaccharides (HMOs. This suggests caprine milk oligosaccharides may offer similar protective and developmental effects to that of HMOs. However, to date, studies using oligosaccharides from caprine milk have been limited. Thus, this study aimed to examine the impact of a caprine milk oligosaccharide-enriched fraction (CMOF on barrier function of epithelial cell co-cultures of absorptive enterocytes (Caco-2 cells and mucus-secreting goblet cells (HT29-MTX cells, that more closely simulate the cell proportions found in the small (90:10 and large intestine (75:25. Treatment of epithelial co-cultures with 0.4, 1.0, 2.0 and 4.0 mg/mL of CMOF was shown to have no effect on metabolic activity but did enhance cell epithelial barrier integrity as measured by trans-epithelial electrical resistance (TEER, in a dose-dependent manner. The CMOF at the maximum concentration tested (4.0 mg/mL enhanced TEER, mucin gene expression and mucin protein abundance of epithelial co-cultures, all of which are essential components of intestinal barrier function.

  9. Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy.

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    Raghav K Chhetri

    Full Text Available The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A or pre-malignant (MCF10DCIS.com MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF. This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios [1] were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled in vitro, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.

  10. Streptococcus mitis/human gingival fibroblasts co-culture: the best natural association in answer to the 2-hydroxyethyl methacrylate release

    Science.gov (United States)

    Di Giulio, Mara; D'Ercole, Simonetta; Zara, Susi; Cataldi, Amelia; Cellini, Luigina

    2012-01-01

    One of the major components of dental polymerized resin-based restorative materials is 2-hydroxyethyl methacrylate (HEMA) and its release in monomeric form interferes with the oral cavity environment. This study aimed to evaluate HEMA monomeric effects on the co-culture of Streptococcus mitis and human gingival fibroblasts (HGFs). Streptococcus mitis DS12 and S. mitis ATCC 6249 were co-cultivated with HGF in the presence of HEMA (3 mM), for 48 and 72 h; the amount of sessile and planktonic cells, as well as the prokaryotic and eukaryotic cell viability were analyzed in treated and untreated samples. The treatment of S. mitis/HGFs with HEMA did not produce significant effects on the bacterial adhesion and induced an increase in planktonic S. mitis ATCC 6249 population after 48 and 72 h. HEMA increased significantly the planktonic S. mitis ATCC 6249 viability when co-cultured with HGFs, while a cytotoxic effect on HGFs, without bacteria, was recorded. An increase of bacterial aggregation on HGFs was also detected with HEMA. Data obtained in this study suggest that HEMA exhibits a toxic effect mainly on eukaryotic cells and this effect can be modulated by co-cultivation with the S. mitis cells which, in the presence of the monomer, enhance their aggregation rate on HGFs. PMID:22229269

  11. Reductive dechlorination of B-hexachlorocyclohexane (B-HCH) by a Dehalobacter species in coculture with a Sedimentibacter species

    NARCIS (Netherlands)

    Doesburg, van W.C.J.; Eekert, van M.H.A.; Middeldorp, P.J.M.; Balk, M.; Schraa, G.; Stams, A.J.M.

    2005-01-01

    An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the ß-HCH isomer to benzene and chlorobenzene in a ratio of 0.5¿2 depending on the amount of ß-HCH degraded. The culture grows with H2 as electron donor and ß-HCH as electron

  12. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis.

    Science.gov (United States)

    Bronckaers, Annelies; Hilkens, Petra; Martens, Wendy; Gervois, Pascal; Ratajczak, Jessica; Struys, Tom; Lambrichts, Ivo

    2014-08-01

    Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Co-culture of microalgae, cyanobacteria, and macromycetes for exopolysaccharides production: process preliminary optimization and partial characterization.

    Science.gov (United States)

    Angelis, S; Novak, A C; Sydney, E B; Soccol, V T; Carvalho, J C; Pandey, A; Noseda, M D; Tholozan, J L; Lorquin, J; Soccol, C R

    2012-07-01

    In this study, the biomass and exopolysaccharides (EPS) production in co-cultures of microalgae/cyanobacteria and macromycetes was evaluated as a technology for producing new polysaccharides for medical and/or industrial application. Based on biomass and EPS productivity of monocultures, two algae and two fungi were selected and cultured in different co-culture arrangements. The hydrosoluble EPS fractions from mono- and co-cultures were characterized by ¹³C NMR spectroscopy and gas chromatography coupled to mass spectrometry and compared. It was found that co-cultures resulted in the production of an EPS different from those produced by monocultures, showing fungal predominance with microalgal/cyanobacterial traces. Co-cultures conditions were screened (temperature, agitation speed, fungal and microalgae inoculation rate, initial pH, illumination rate, and glucose concentration) in order to achieve maximum biomass and EPS production, resulting in an increase of 33 and 61% in exopolysaccharides and biomass productions, respectively (patent pending).

  14. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli.

    Science.gov (United States)

    Zhang, Haoran; Stephanopoulos, Gregory

    2016-07-01

    3-amino-benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli-E. coli co-culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co-culture system was found to improve 3AB production by 15 fold, compared to the mono-culture approach. Further engineering of the co-culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co-culture engineering can be a powerful new approach in the broad field of metabolic engineering. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Boosting dark fermentation with co-cultures of extreme thermophiles for biohythane production from garden waste.

    Science.gov (United States)

    Abreu, Angela A; Tavares, Fábio; Alves, Maria Madalena; Pereira, Maria Alcina

    2016-11-01

    Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS). Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Ethanol production from corn cobs by co-culture of Saccharomyces ...

    African Journals Online (AJOL)

    Saccharomyces cerevisiae and Aspergillus niger were used in a co-culture for the simultaneous saccharification and fermentation (SSF) of 1% and 10% (w/v) dry pre-treated corn cobs to ethanol. Positive controls of glucose of same concentrations in a synthetic medium were also fermented. At 1% substrate concentration, ...

  17. The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production.

    Science.gov (United States)

    Caron-Beaudoin, Elyse; Viau, Rachel; Hudon-Thibeault, Andrée-Anne; Vaillancourt, Cathy; Sanderson, J Thomas

    2017-10-01

    Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Treatment of real wastewater using co-culture of immobilized Chlorella vulgaris and suspended activated sludge.

    Science.gov (United States)

    Mujtaba, Ghulam; Lee, Kisay

    2017-09-01

    The use of algal-bacterial symbiotic association establishes a sustainable and cost-effective strategy in wastewater treatment. Using municipal wastewater, the removal performances of inorganic nutrients (nitrogen and phosphorus) and organic pollutants were investigated by the co-culture system having different inoculum ratios (R) of suspended activated sludge to alginate-immobilized microalgae Chlorella vulgaris. The co-culture reactors with lower R ratios obtained more removal of nitrogen than in pure culture of C. vulgaris. The reactor with R = 0.5 (sludge/microalgae) showed the highest performance representing 66% removal after 24 h and 95% removal after 84 h. Phosphorus was completely eliminated (100%) in the co-culture system with inoculum ratios of 0.5 and 1.0 after 24 h and in the pure C. vulgaris culture after 36 h. The COD level was greatly reduced in the activated sludge reactor, while, it was increasing in pure C. vulgaris culture after 24 h of incubation. However, COD was almost stabilized after 24 h in the reactors with high R ratios such as 2.0, 5.0, and 10 due to the higher concentration of activated sludge. The growth of C. vulgaris was promoted from 0.03 g/L/d to 0.05 g/L/d in the co-culture of low inoculum ratios such as R = 0.5, implying that there exist an optimum inoculum ratio in the co-culture system in order to achieve efficient removal of nutrients. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. [Fluid suspension co-culture of Dendrobium officinale protocorm and living fungus].

    Science.gov (United States)

    Meng, Zhixia; Shu, Ying; Wang, Chunlan; Guo, Shunxing

    2012-06-01

    To set up a system for fluid suspension co-culture of Dendrobium officinale protocorm and living fungus MF24, so as to provide certain scientific evidence for industrial production of protocorm. Whether the protocorm culture system was suitable for the normal growth of MF24 fungus were studied, the growth of protocorm cultured alone and co-cultured with the fungus were researched under light and dark culture conditions, the biomass and proliferation times were determined, and HPLC method was used to analyze and compare the changes of 11 characteristic peak areas in D. officinale protocorm. The MF24 fungus could grow normally in the 6,7-V liquid medium used to culture the protocorm, and when it was cultured by 8-10 hours per day under 1 500 lx, the growth rate of the fungus was slowed. Protocorm could grow normally in light and dark culture conditions, and add the MF24 fungus in the early cultivation stages of protocorm, both inhibit the growth of each other. In the protocorm for the growth stability to add 5 diameter 9 mm fungi block, the protocorm growth and chemical composition type had no significant effect. However, under illumination, co-cultured for 5 days protocorm of which 10 compounds content decreased 13.64% to 138.47%, in dark conditions, co-cultured for 5 days protocorm of which 7 compounds increased by 0.71% to 12.82%, and 4 compounds slightly reduced by 3.03% to 14. 14% compared with the control. Under the appropriate condition, living fungus MF24 could co-culture with the D. officinale protocorm, and affected the latter's secondary metabolite levels.

  20. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Qingbei Zhang

    Full Text Available Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3 and basal (MDA-MB-231 types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL and glioblastoma cell lines (U87 and D54. These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

  1. Impact of coculturing Bifidobacterium animalis subsp. lactis HN019 with yeasts on microbial viability and metabolite formation.

    Science.gov (United States)

    Toh, M; Liu, S-Q

    2017-10-01

    To evaluate the impact of coculturing Bifidobacterium animalis subsp. lactis HN019 with yeasts on microbial viability and metabolite production. Monocultures and bacteria-yeast cocultures of B. lactis HN019 and 10 different yeast strains belonging to different species in skim milk media were fermented at 37°C. The presence of yeasts enhanced the growth rate and metabolic activities of B. lactis HN019, which might be attributed to their antioxidative properties. The viability of yeasts, when cocultured with bifidobacteria, was either unaffected or suppressed, depending on the strain. When the B. lactis HN019 monoculture and cocultures with Saccharomyces cerevisiae EC-1118, Pichia kluyveri FrootZen and Kluyveromyces lactis KL71 were fermented to pH 4·7, there were no significant differences in their organic acid composition. On the other hand, cocultures produced significantly higher quantities of alcohols and/or esters than the monoculture. Coculturing B. lactis HN019 with yeasts did not improve the viability of the probiotic during storage at 10°C for 8 weeks, as the bifidobacteria itself demonstrated satisfactory survival in the fermented SMM. Coculturing B. lactis HN019 with yeasts accelerated the growth of the bifidobacteria and increased the production aroma-active volatile metabolites. This study demonstrates the potential of utilizing specific yeast species as starter or adjunct cultures to simultaneously improve the growth of fastidious bifidobacteria and modulate the organoleptic properties of fermented food products. © 2017 The Society for Applied Microbiology.

  2. Polarized Airway Epithelial Models for Immunological Co-Culture Studies

    DEFF Research Database (Denmark)

    Papazian, Dick; Würtzen, Peter A; Hansen, Søren Werner Karlskov

    2016-01-01

    of the epithelial response towards environmental antigens and genetic susceptibility, resulting in inflammation and T cell-derived immune responses. In vivo animal models have long been used to study immune homeostasis of the airways but are limited by species restriction and lack of exposure to a natural......Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation......-culture models to become powerful tools in the discovery of key molecules dictating immunity and/or tolerance, and for understanding the complex interplay that takes place between mucosa, airway epithelium and resident or infiltrating immune cells. This review focuses on current knowledge and the advantages...

  3. Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte-HaCaT keratinocyte co-culture system in vitro.

    Science.gov (United States)

    Ma, Hui-Jun; Zhao, Guang; Zi, Shao-Xia; Li, Dong-Guang; Liu, Wen; Yang, Qing-Qi

    2010-08-01

    In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co-culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co-cultured for 72 h in a serum-free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)-conjugated antibody against the melanosome-specific protein gp100, and with Phycoerythrin (PE)-conjugated antibody against the keratinocyte-specific marker cytokeratin. Then, the cells were examined using co-focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time-dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, alpha-melanocyte stimulating hormone (alpha-MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose-dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer.

  4. In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

    International Nuclear Information System (INIS)

    Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

    2005-01-01

    It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

  5. Engineering Escherichia coli coculture systems for the production of biochemical products.

    Science.gov (United States)

    Zhang, Haoran; Pereira, Brian; Li, Zhengjun; Stephanopoulos, Gregory

    2015-07-07

    Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.

  6. Increased methane production in cyanobacteria and methanogenic microbe co-cultures.

    Science.gov (United States)

    Yeung, Tracey; Kwan, Matthew; Adler, Lewis; Mills, Toby J; Neilan, Brett A; Conibeer, Gavin; Patterson, Robert

    2017-11-01

    A novel light-to-bioenergy system produced 3.5 times the baseline methane output using a co-culture of cyanobacteria (Oscillatoria sp.) and a methanogenic microbial community. Analysis of micronutrients in the system during the growth phase indicated that cobalt, iron, nickel and zinc were not appreciably consumed. The stable consumption and return of macronutrients calcium and magnesium were also observed. Essential macronutrients nitrogen, in the form of nitrate, and phosphorus showed no cycling during the growth phase and were depleted at rates of 0.35mg/L/day and 0.40µg/L/day, respectively. Biofilm formation increased the resilience of biomass to bacterial degradation in an anaerobic digester, as shown by viability assays of cyanobacterial biofilms in the co-culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Interspecies cross-talk between co-cultured Pseudomonas putida and Escherichia coli.

    Science.gov (United States)

    Molina-Santiago, Carlos; Udaondo, Zulema; Cordero, Baldo F; Ramos, Juan L

    2017-08-01

    Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be isolated from soil rhizosphere, the surface of vegetables, fresh waters and wastewaters - environments in which they likely co-exist. Despite this, the potential interactions between these microbes have not been studied in detail. To analyse these interactions, we carried out RNA-seq transcriptomic analysis of these microbes as monocultures and as co-cultures. Our results show that co-culture of these microbes significantly alters transcriptional profiles. The most dramatic transcriptional changes in both microorganisms were involved in central carbon metabolism, as well as adhesion to surfaces and the activation of drug efflux pumps. We also found that acetate production was one of the mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida DOT-T1E. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Dopamine D1 receptor activation regulates the expression of the estrogen synthesis gene aromatase B in radial glial cell

    Directory of Open Access Journals (Sweden)

    Lei eXing

    2015-09-01

    Full Text Available Radial glial cells (RGCs are abundant stem-like non-neuronal progenitors that are important for adult neurogenesis and brain repair, yet little is known about their regulation by neurotransmitters. Here we provide evidence for neuronal-glial interactions via a novel role for dopamine to stimulate RGC function. Goldfish were chosen as the model organism due to the abundance of RGCs and regenerative abilities of the adult central nervous system. A close anatomical relationship was observed between tyrosine hydroxylase-positive catecholaminergic cell bodies and axons and dopamine-D1 receptor expressing RGCs along the ventricular surface of telencephalon, a site of active neurogenesis. A primary cell culture model was established and immunofluorescence analysis indicates that in vitro RGCs from female goldfish retain their major characteristics in vivo, including expression of glial fibrillary acidic protein and brain lipid binding protein. The estrogen synthesis enzyme aromatase B is exclusively found in RGCs, but this is lost as cells differentiate to neurons and other glial types in adult teleost brain. Pharmacological experiments using the cultured RGCs established that specific activation of dopamine D1 receptors up-regulates aromatase B mRNA through a cyclic adenosine monophosphate-dependent molecular mechanism. These data indicate that dopamine enhances the steroidogenic function of this neuronal progenitor cell.

  9. Assessment of a micropatterned hepatocyte coculture system to generate major human excretory and circulating drug metabolites.

    Science.gov (United States)

    Wang, Wendy WeiWei; Khetani, Salman R; Krzyzewski, Stacy; Duignan, David B; Obach, R Scott

    2010-10-01

    Metabolism is one of the important determinants of the overall disposition of drugs, and the profile of metabolites can have an impact on efficacy and safety. Predicting which drug metabolites will be quantitatively predominant in humans has become increasingly important in the research and development of new drugs. In this study, a novel micropatterned hepatocyte coculture system was evaluated for its ability to generate human in vivo metabolites. Twenty-seven compounds of diverse chemical structure and subject to a range of drug biotransformation reactions were assessed for metabolite profiles in the micropatterned coculture system using pooled cryopreserved human hepatocytes. The ability of this system to generate metabolites that are >10% of dose in excreta or >10% of total drug-related material in circulation was assessed and compared to previously reported data obtained in human hepatocyte suspensions, liver S-9 fraction, and liver microsomes. The micropatterned coculture system was incubated for up to 7 days without a change in medium, which offered an ability to generate metabolites for slowly metabolized compounds. The micropatterned coculture system generated 82% of the excretory metabolites that exceed 10% of dose and 75% of the circulating metabolites that exceed 10% of total circulating drug-related material, exceeds the performance of hepatocyte suspension incubations and other in vitro systems. Phase 1 and phase 2 metabolites were generated, as well as metabolites that arise via two or more sequential reactions. These results suggest that this in vitro system offers the highest performance among in vitro metabolism systems to predict major human in vivo metabolites.

  10. The secretome signature of reactive glial cells and its pathological implications.

    Science.gov (United States)

    Jha, Mithilesh Kumar; Seo, Minchul; Kim, Jong-Heon; Kim, Byung-Gyu; Cho, Je-Yoel; Suk, Kyoungho

    2013-11-01

    Glial cells are non-neuronal components of the central nervous system (CNS). They are endowed with diverse functions and are provided with tools to detect their own activities and those of neighboring neurons. Glia and neurons are in continuous reciprocal communication under both physiological and neuropathological conditions, and glia secrete various guidance factors or proteinaceous signals that service vital neuronal-glial interactions in health and disease. Analysis and profiling of glial secretome, especially of microglia and astrocytes, have raised new expectations for the diagnosis and treatment of CNS disorders, and the availability of a catalog of glia-secreted proteins might provide an origin for further research on the complex extracellular signaling mediated by glial cells. Components of the glial secretome play important roles as mediators and modulators of brain structure and function during neuroprotection and neurodegeneration. Therapeutic hypothermia has been acclaimed an effective modulator of brain injury via its substantial effect on the protein expression profiles of glia. Furthermore, emerging proteomic tools and methodologies make feasible the documentation of the reactive glial secretome signature. This review focuses on reactive glial cells and the uniqueness of their secretome during diverse neuropathological conditions. This article is part of a Special Issue entitled: An Updated Secretome. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Absorption and Metabolism of Phenolics from Digests of Polyphenol-Rich Potato Extracts Using the Caco-2/HepG2 Co-Culture System.

    Science.gov (United States)

    Sadeghi Ekbatan, Shima; Iskandar, Michele M; Sleno, Lekha; Sabally, Kebba; Khairallah, Joelle; Prakash, Satya; Kubow, Stan

    2018-01-12

    The bioactivity of dietary polyphenols depends upon gastrointestinal and hepatic metabolism of secondary microbial phenolic metabolites generated via colonic microbiota-mediated biotransformation. A polyphenol-rich potato extract (PRPE) containing chlorogenic, caffeic, and ferulic acids and rutin was digested in a dynamic multi-reactor gastrointestinal simulator of the human intestinal microbial ecosystem (GI model). Simulated digestion showed extensive degradation of the parent compounds and the generation of microbial phenolic metabolites. To characterize the transport and metabolism of microbial phenolic metabolites following digestion, a co-culture of intestinal Caco-2 and hepatic HepG2 cells was exposed to the PRPE-derived digests obtained from the colonic vessels. Following a 2 h incubation of the digesta with the Caco-2/HepG2 co-cultures, approximately 10-15% of ferulic, dihydrocaffeic, and dihydroferulic acids and 3-5% of 3-hydroxybenzoic, 3-hydroxyphenylpropionic, and coumaric acids were observed in the basolateral side, whereas 3-hydroxyphenylacetic acid, phenylpropanoic acid, and cinnamic acid were not detected. Subsequent HepG2 cellular metabolism led to major increases in ferulic, dihydrocaffeic, 3-hydroxyphenylpropionic, and coumaric acids ranging from 160-370%. These findings highlight the importance of hepatic metabolism towards the generation of secondary metabolites of polyphenols despite low selective Caco-2 cellular uptake of microbial phenolic metabolites.

  12. Absorption and Metabolism of Phenolics from Digests of Polyphenol-Rich Potato Extracts Using the Caco-2/HepG2 Co-Culture System

    Directory of Open Access Journals (Sweden)

    Shima Sadeghi Ekbatan

    2018-01-01

    Full Text Available The bioactivity of dietary polyphenols depends upon gastrointestinal and hepatic metabolism of secondary microbial phenolic metabolites generated via colonic microbiota-mediated biotransformation. A polyphenol-rich potato extract (PRPE containing chlorogenic, caffeic, and ferulic acids and rutin was digested in a dynamic multi-reactor gastrointestinal simulator of the human intestinal microbial ecosystem (GI model. Simulated digestion showed extensive degradation of the parent compounds and the generation of microbial phenolic metabolites. To characterize the transport and metabolism of microbial phenolic metabolites following digestion, a co-culture of intestinal Caco-2 and hepatic HepG2 cells was exposed to the PRPE-derived digests obtained from the colonic vessels. Following a 2 h incubation of the digesta with the Caco-2/HepG2 co-cultures, approximately 10–15% of ferulic, dihydrocaffeic, and dihydroferulic acids and 3–5% of 3-hydroxybenzoic, 3-hydroxyphenylpropionic, and coumaric acids were observed in the basolateral side, whereas 3-hydroxyphenylacetic acid, phenylpropanoic acid, and cinnamic acid were not detected. Subsequent HepG2 cellular metabolism led to major increases in ferulic, dihydrocaffeic, 3-hydroxyphenylpropionic, and coumaric acids ranging from 160–370%. These findings highlight the importance of hepatic metabolism towards the generation of secondary metabolites of polyphenols despite low selective Caco-2 cellular uptake of microbial phenolic metabolites.

  13. Human umbilical cord-derived mesenchymal stem cells inhibit proliferation but maintain survival of Jurkat leukemia cells in vitro by activating Notch signaling.

    Science.gov (United States)

    Yuan, Yin; Chen, Danliang; Chen, Xuan; Shao, Hongwei; Huang, Shulin

    2014-04-01

    To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the proliferation and survival of Jurkat leukemia cells in vitro and explore the possible mechanism. Jurkat leukemia cells were co-cultured with hUC-MSCs isolated from human umbilical cord tissues by plastic adherence at a ratio of 10:1. The proliferation and survival of the co-cultured Jurkat cells, separated by immunomagnetic bead cell sorting on day 4, were evaluated by flow cytometry. Western blotting was performed to evaluate the activation of Notch signaling in the co-cultured Jurkat cells. Jurkat leukemia cells co-cultured with hUC-MSCs for 4 days showed a lowered proliferation rate and cell cycle arrest at G0/G1 phase with a reduction in the cell apoptotic rate. Notch signaling pathway was activated in the co-cultured Jurkat cells as evidenced by an increased cellular expression of HES-1. Co-culture with hUC-MSCs can inhibit the proliferation of Jurkat leukemia cells in vitro and protect the cells from apoptosis by activating Notch signaling, indicating a potential shielding effect of MSCs on leukemia cells.

  14. Optical imaging of three dimensional cell constructs grown in microgravity and in immunodeficient mice, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Rolling wall vessels (RWV) utilizing both single cell lines and co-cultures with two or more cell types are contributing significantly to our understanding of tumor...

  15. Optical imaging of three dimensional cell constructs grown in microgravity and in immunodeficient mice Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Rolling wall vessels (RWV) utilizing both single cell lines and co-cultures with two or more cell types are contributing significantly to our understanding of tumor...

  16. Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats.

    Science.gov (United States)

    Gwak, Young S; Kang, Jonghoon; Unabia, Geda C; Hulsebosch, Claire E

    2012-04-01

    In the spinal cord, neuron and glial cells actively interact and contribute to neurofunction. Surprisingly, both cell types have similar receptors, transporters and ion channels and also produce similar neurotransmitters and cytokines. The neuroanatomical and neurochemical similarities work synergistically to maintain physiological homeostasis in the normal spinal cord. However, in trauma or disease states, spinal glia become activated, dorsal horn neurons become hyperexcitable contributing to sensitized neuronal-glial circuits. The maladaptive spinal circuits directly affect synaptic excitability, including activation of intracellular downstream cascades that result in enhanced evoked and spontaneous activity in dorsal horn neurons with the result that abnormal pain syndromes develop. Recent literature reported that spinal cord injury produces glial activation in the dorsal horn; however, the majority of glial activation studies after SCI have focused on transient and/or acute time points, from a few hours to 1 month, and peri-lesion sites, a few millimeters rostral and caudal to the lesion site. In addition, thoracic spinal cord injury produces activation of astrocytes and microglia that contributes to dorsal horn neuronal hyperexcitability and central neuropathic pain in above-level, at-level and below-level segments remote from the lesion in the spinal cord. The cellular and molecular events of glial activation are not simple events, rather they are the consequence of a combination of several neurochemical and neurophysiological changes following SCI. The ionic imbalances, neuroinflammation and alterations of cell cycle proteins after SCI are predominant components for neuroanatomical and neurochemical changes that result in glial activation. More importantly, SCI induced release of glutamate, proinflammatory cytokines, ATP, reactive oxygen species (ROS) and neurotrophic factors trigger activation of postsynaptic neuron and glial cells via their own receptors

  17. Spatial and Temporal Activation of Spinal Glial Cells: Role of Gliopathy in Central Neuropathic Pain Following Spinal Cord Injury in Rats

    Science.gov (United States)

    Gwak, Young S.; Kang, Jonghoon; Unabia, Geda C.; Hulsebosch, Claire. E.

    2012-01-01

    In the spinal cord, neurons and glial cells actively interact and contribute to neurofunction. Surprisingly, both cell types have similar receptors, transporters and ion channels and also produce similar neurotransmitters and cytokines. The neuroanatomical and neurochemical similarities work synergistically to maintain physiological homeostasis in the normal spinal cord. However, in trauma or disease states, spinal glia become activated, dorsal horn neurons become hyperexcitable contributing to sensitized neuronal-glial circuits. The maladaptive spinal circuits directly affect synaptic excitability, including activation of intracellular downstream cascades that result in enhanced evoked and spontaneous activity in dorsal horn neurons with the result that abnormal pain syndromes develop. Recent literature reported that spinal cord injury produces glial activation in the dorsal horn; however, the majority of glial activation studies after SCI have focused on transient and/or acute time points, from a few hours to one month, and peri-lesion sites, a few millimeters rostral and caudal to the lesion site. In addition, thoracic spinal cord injury produces activation of astrocytes and microglia that contributes to dorsal horn neuronal hyperexcitability and central neuropathic pain in above-level, at-level and below-level segments remote from the lesion in the spinal cord. The cellular and molecular events of glial activation are not a simple event, rather it is the consequence of a combination of several neurochemical and neurophysiological changes following SCI. The ionic imbalances, neuroinflammation and alterations of cell cycle proteins after SCI are predominant components for neuroanatomical and neurochemical changes that result in glial activation. More importantly, SCI induced release of glutamate, proinfloammatory cytokines, ATP, reactive oxygen species (ROS) and neurotrophic factors trigger activation of postsynaptic neurons and glial cells via their own

  18. Sparse and Specific Coding during Information Transmission between Co-cultured Dentate Gyrus and CA3 Hippocampal Networks.

    Science.gov (United States)

    Poli, Daniele; Thiagarajan, Srikanth; DeMarse, Thomas B; Wheeler, Bruce C; Brewer, Gregory J

    2017-01-01

    To better understand encoding and decoding of stimulus information in two specific hippocampal sub-regions, we isolated and co-cultured rat primary dentate gyrus (DG) and CA3 neurons within a two-chamber device with axonal connectivity via micro-tunnels. We tested the hypothesis that, in these engineered networks, decoding performance of stimulus site information would be more accurate when stimuli and information flow occur in anatomically correct feed-forward DG to CA3 vs. CA3 back to DG. In particular, we characterized the neural code of these sub-regions by measuring sparseness and uniqueness of the responses evoked by specific paired-pulse stimuli. We used the evoked responses in CA3 to decode the stimulation sites in DG (and vice-versa) by means of learning algorithms for classification (support vector machine, SVM). The device was placed over an 8 × 8 grid of extracellular electrodes (micro-electrode array, MEA) in order to provide a platform for monitoring development, self-organization, and improved access to stimulation and recording at multiple sites. The micro-tunnels were designed with dimensions 3 × 10 × 400 μm allowing axonal growth but not migration of cell bodies and long enough to exclude traversal by dendrites. Paired-pulse stimulation (inter-pulse interval 50 ms) was applied at 22 different sites and repeated 25 times in each chamber for each sub-region to evoke time-locked activity. DG-DG and CA3-CA3 networks were used as controls. Stimulation in DG drove signals through the axons in the tunnels to activate a relatively small set of specific electrodes in CA3 (sparse code). CA3-CA3 and DG-DG controls were less sparse in coding than CA3 in DG-CA3 networks. Using all target electrodes with the three highest spike rates (14%), the evoked responses in CA3 specified each stimulation site in DG with optimum uniqueness of 64%. Finally, by SVM learning, these evoked responses in CA3 correctly decoded the stimulation sites in DG for 43% of the

  19. Pseudomonads Isolated from Pristine Background Groundwater Proliferate More Effectively in Co-culture than in Monoculture Under Denitrifying Conditions

    Science.gov (United States)

    Aaring, A. B.; Lancaster, A.; Novichkov, P.; Adams, M. W. W.; Deutschbauer, A. M.; Chakraborty, R.

    2016-12-01

    As part of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) consortium, we study the microbial community at the U.S. Department of Energy's Field Research Center (FRC) in Oak Ridge. The groundwater at this site contains plumes of nitrate with concentrations up to 14,000mg/L among other contaminants, though molybdenum concentrations are low. Because molybdenum is essential to nitrate reduction, this can be inhibitory to growth. Several strains of Pseudomonas were isolated from the same background groundwater sample. These isolates utilized diverse carbon sources ranging from acetate to glucose while growing under denitrifying conditions. The strains were also screened for nitrate tolerance and a couple of them were shown to be tolerant to 300-400 mM nitrate under anaerobic conditions. In the field site the bacteria live in consortia rather than in isolation, therefore we hypothesized that growth of these strains will be more robust in co-culture, as the denitrification pathway was segmented between the species. Three of the isolates (Pseudomonas fluorescens strains N1B4, N2E2, N2E3) were selected for in-depth analysis based on growth in pairwise co-cultures relative to monocultures, and the availability of the relevant genetic tools, such as transposon mutant libraries. Full genome sequencing showed that strain N2E3 has a truncated dentrification pathway: it lacks nitrous oxide reductase. Our results show strain N2E2 grow to maximum cell density an average of 45 hours more quickly when grown with strain N2E3 than in monoculture. Utilizing RB-TnSeq libraries of our strains, it was also found that some genes involved in nitrate reduction, sulfate permeability, molybdenum utilization, and anaerobic reduction are important for growth under these conditions. In addition, a few unexpected genes were also shown to be positively correlated to growth, such as genes homologous to genes for DNA proofreading or antibiotic production. These

  20. Initial Attempts of Development and Characterization of an In Vitro Blood Brain Barrier Model Derived from Human Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Hall, Vanessa Jane

    observed for several days after seeding by measuring the trans-endothelial electrical resistance (TEER). Initial pilot studies have shown a significant difference between stem cells grown as a mono culture and stem cells grown in co-culture with rat astrocytes. The stem cell line WTSli024-A had...... configurations (mono culture, non-contact co-culture and contact co-culture) with primary rat astrocytes to induce barrier-like properties. Endothelial cell media supplemented with retinoic acid were then applied to the cells to ensure selective expansion of BECs. The different culture configurations were...