Sample records for brain cell cultures

  1. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya


    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions....

  2. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro culturing......The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...... and electrochemical sensor system that enables real time detection of metabolites, e.g. dopamine from cell cultures and brain slices. In summary we present results on culturing of brain slices and cells in the microfluidic system as well as on the incorporation of an electrochemical sensor system for characterization...

  3. 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


    In vitro models of the blood-brain barrier are useful tools to study blood-brain barrier function as well as drug permeation from the systemic circulation to the brain parenchyma. However, a large number of the available in vitro models fail to reflect the tightness of the in vivo blood-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....

  4. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E


    of gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced...... morphological differentiation of epitheloid neural cells into astrocytes. This occurred in carcinogen treated cells as well as in untreated control cultures. At the same time cells with astrocyte morphology showed accumulation of glial fibrillary acidic protein (GFA) as tested by indirect immunofluorescence...... with monospecific antibodies against GFA. Thereafter, in the EtNU pre-treated cultures an increased number of cells with astrocyte morphology was seen, and BE further increased the number of cells with long cytoplasmic processes. Control cells were GFA negative, while some few strongly, as well as many weakly...

  5. Cultured human embryonic neocortical cells survive and grow in infarcted cavities of adult rat brains and interconnect with host brain

    Institute of Scientific and Technical Information of China (English)

    ZENG Jin-sheng; YU Jian; CUI Chun-mei; ZHAO Zhan; HONG Hua; SHENG Wen-li; TAO Yu-qian; LI Ling; HUANG Ru-xun


    Background There are no reports on exnografting cultured human fetal neocortical cells in this infracted cavities of adult rat brains. This study was undertaken to observe whether cultured human cortical neurons and astrocytes can survive and grow in the infarcted cavities of adult rat brains and whether they interconnect with host brains.Methods The right middle cerebral artery was ligated distal to the striatal branches in 16 adult stroke-prone renovascular hypertensive rats. One week later, cultured cells from human embryonic cerebral cortexes were stereotaxically transferred to the infarcted cavity of 11 rats. The other 5 rats receiving sham transplants served as controls. For immunosuppression, all transplanted rats received intraperitoneal injection of cyclosporine A daily starting on the day of grafting. Immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin, neurofilament, and microtubule associated protein-2 (MAP-2) was performed on brain sections perfused in situ 8 weeks after transplantation.Results Grafts in the infarcted cavities of 6 of 10 surviving rats consisted of bands of neurons with an immature appearance, bundles of fibers, and GFAP-immunopositive astrocytes, which were unevenly distributed. The grafts were rich in synaptophysin, neurofilament, and MAP2-positive neurons with long processes. The graft/host border was diffuse with dendrites apparently bridging over to the host brain, into which neurofilament immunopositive fibers protruded. Conclusion Cultured human fetal brain cells can survive and grow in the infarcted cavities of immunodepressed rats and integrate with the host brain.

  6. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells. (United States)

    Parkinson, Fiona E; Hacking, Cindy


    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  7. Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects. (United States)

    Wilson, Daniel E; Velarde, Rodrigo A; Fahrbach, Susan E; Mommaerts, Veerle; Smagghe, Guy


    Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

  8. Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases. (United States)

    Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J


    Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases.

  9. Effects of heavy ion to the primary culture of mouse brain cells (United States)

    Nojima, Kumie; Nakadai, Taeko; Kohno, Yukio; Vazquez, Marcelo E.; Yasuda, Nakahiro; Nagaoka, Shunji


    To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.

  10. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyoshi, Ko, E-mail: [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)


    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  11. Brain, body and culture

    DEFF Research Database (Denmark)

    Geertz, Armin W.


    This essay sketches out a biocultural theory of religion which is based on an expanded view of cognition that is anchored in brain and body (embrained and embodied), deeply dependent on culture (enculturated) and extended and distributed beyond the borders of individual brains. Such an approach...... uniquely accommodates contemporary cultural and neurobiological sciences. Since the challenge that the study of religion faces, in my opinion, is at the interstices of these sciences, I have tried to develop a theory of religion which acknowledges the fact. My hope is that the theory can be of use...

  12. Ultrastructural identification of Ricinus communis agglutinin-1 positive cells in primary dissociated cell cultures of human embryonic brain. (United States)

    Bobryshev, Y; Ashwell, K


    While Ricinus communis agglutinin 1 (RCA-1) can be used as a specific marker to study the development and differentiation of microglial cells in human embryogenesis, little is known about the structural heterogeneity and nature of RCA-1+ cells. To analyse the structural peculiarities of RCA-1+ cells, we have used primary dissociated cultures of human embryonic brain. These have been used as models for investigating many of the aspects of central nervous system (CNS) HIV infection. We have shown that primary dissociated cultures from human embryos as young as 10 weeks gestation contain RCA-1+ cells. The RCA-1+ cells exist in two forms, those without (type I) and those with (type II) processes. The former have a poorly developed ultrastructure, while the latter have well developed ultrastructural features, such as rough endoplasmic reticulum with short cisternae, abundant ribosomes, mitochondria, lysosomes and vacuoles. Furthermore, some of these cells with processes have well developed cytoskeletal features. In this paper, the classification of RCA-1+ cells of embryonic human brain is considered and their morphology compared to microglia identified in rodent CNS.

  13. Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment

    Institute of Scientific and Technical Information of China (English)

    Xianying Wang; Honghai Wu; Gai Xue; Yanning Hou


    In this study, human umbilical cord mesenchymal stem cells from full-term neonates born by vaginal delivery were cultured in medium containing 150 mg/mL of brain tissue extracts from Sprague-Dawley rats (to mimic the brain microenvironment). Immunocytochemical analysis demonstrated that the cells differentiated into neuron-like cells. To evaluate the effects of progesterone as a neurosteroid on the neuronal differentiation of human umbilical cord mesenchymal stem cells, we cultured the cells in medium containing progesterone (0.1, 1, 10 μM) in addition to brain tissue extracts. Reverse transcription-PCR and flow cytometric analysis of neuron specific enolase-positive cells revealed that the percentages of these cells increased significantly following progesterone treatment, with the optimal progesterone concentration for neuron-like differentiation being 1 μM. These results suggest that progesterone can enhance the neuronal differentiation of human umbilical cord mesenchymal stem cells in culture medium containing brain tissue extracts to mimic the brain microenvironment.

  14. Direct Signaling from Astrocytes to Neurons in Cultures of Mammalian Brain Cells (United States)

    Nedergaard, Maiken


    Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

  15. Pharmacological assessment of ARTCEREB irrigation and perfusion solution for cerebrospinal surgery using primary cultures of rat brain cells. (United States)

    Nishimura, Masuhiro; Doi, Kazuhisa; Kishimoto, Sanae; Koshitani, Osamu; Naito, Shinsaku; Yamauchi, Aiko


    ARTCEREB irrigation and perfusion solution (Artcereb), an ethical pharmaceutical, is typically applied inside the skull and spinal cavity as artificial fluid. Artcereb is composed of glucose and electrolytes (Na+, K+, Mg2+, Ca2+, Cl-, HCO3- and P) and has a pH of 7.3. An in vitro assessment of the effects of Artcereb on cell culture of rat fetal astrocytes or rat fetal brain cells was performed in comparison with normal saline and lactated Ringer's solutions. Furthermore, the effects of Artcereb on cell culture of rat fetal brain cells were also assessed in comparison with Krebs bicarbonate solution. Cell function after exposure to Artcereb was assessed based on 3H-thymidine incorporation activity. Cell function after exposure to Artcereb and lactated Ringer's solution in primary cultures of rat fetal astrocytes remained unaffected when compared to that after exposure to normal saline. Cell function after exposure to Artcereb in a primary culture of rat brain cells remained unaffected as compared to that after exposure to normal saline and lactated Ringer's solution. However, function decreased after exposure to a modified Artcereb formulation lacking bicarbonate, thus confirming that the presence of bicarbonate is essential for the Artcereb formulation.

  16. Oxidative Stress Induced by Cigarette Smoke Extracts in Human Brain Cells (T98G) and Human Brain Microvascular Endothelial Cells (HBMEC) in Mono- and Co-Culture. (United States)

    Kim, Ju-Hyeong; Cho, Myung-Haing; Choi, Kyung-Chul; Lee, Kyuhong; Kim, Kwang-Sik; Shim, Soon-Mi


    The objective of the current study was to examine oxidative stress induced by cigarette smoke extract (CSE) or cigarette smoke condensate (CSC) in human brain cells (T98G) and human brain microvascular endothelial cells (HBMEC) in mono- and co-culture systems. Cell viability of T98G cells exposed to CSC (0.05-4 mg/ml) was significantly decreased compared to CSE (0.025-20%). There were no marked differences between quantities of reactive oxygen species (ROS) generation by either CSE (2, 4, and 10%) or CSC (0.2, 0.4, and 0.8 mg/ml) treatment compared to control. However, a significant effect was noted in ROS generation following CSC incubation at 4mg/ml. Cellular integrity of HBMEC decreased to 74 and 64% within 120 h of exposure at the IC50 value of CSE and CSC, respectively. This study suggests that chronic exposure to cigarette smoking might initiate damage to the blood-brain barrier.

  17. Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue



    Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain inj...

  18. Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures. (United States)

    Mao, Xiaoou; Xie, Lin; Greenberg, David A


    Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.

  19. Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum-free conditions (United States)

    Vik-Mo, Einar Osland; Sandberg, Cecilie; Olstorn, Havard; Varghese, Mercy; Brandal, Petter; Ramm-Pettersen, Jon; Murrell, Wayne; Langmoen, Iver Arne


    Traditional in vitro culturing of tumor cells has been shown to induce changes so that cultures no longer represent the tumor of origin. Serum-free culturing conditions are used in a variety of cancers to propagate stem-like cells in vitro. Limited reports, however, exist on the effects of such propagation. We have compared cells from brain tumor biopsies cultivated under serum-free conditions at passages 2 and 10 to describe the effects of in vitro culturing. We were able to establish cell lines from 7 of 10 biopsies from patients with glioblastoma. The cell lines adapted to conditions and had 2.2 times increased population doubling rate at later passages. Karyotyping and comparative genomic hybridization analysis revealed that all examined cell lines had cytogenetic aberrations commonly found in glioblastomas, and there were only minor differences between tumor and early and late passages in the same culture. Whole-transcriptome analysis shows that tumors had interindividual differences. Changes in the overall expression patterns through passaging were modest, with a significant change in only 14 genes; the variation among cultures was, however, reduced through passages. The ability to differentiate differed among tumors but was maintained throughout passaging. The cells initiated tumors upon transplantation to immunodeficient mice with differing phenotypes, but a given cell culture maintained tumor phenotype after serial cultivation. The cultures established maintained individual characteristics specific to culture identity. Thus, each cell culture reflects an image of the tumor—or a personalized model—from which it was derived and remains representative after moderate expansion. PMID:20843775

  20. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model. (United States)

    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y


    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  1. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

    DEFF Research Database (Denmark)

    Schou-Pedersen, Anne Marie Voigt; Hansen, Stine Normann; Tveden-Nyborg, Pernille


    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical...

  2. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells. (United States)

    Minami, Haruka; Tashiro, Katsuhisa; Okada, Atsumasa; Hirata, Nobue; Yamaguchi, Tomoko; Takayama, Kazuo; Mizuguchi, Hiroyuki; Kawabata, Kenji


    The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

  3. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells.

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    Haruka Minami

    Full Text Available The blood brain barrier (BBB is formed by brain microvascular endothelial cells (BMECs and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs were differentiated into endothelial cells (ECs, and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM, in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.

  4. Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

    Directory of Open Access Journals (Sweden)

    Olga M. Pulido


    Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

  5. Apparent reduction of ADAM10 in scrapie-infected cultured cells and in the brains of scrapie-infected rodents. (United States)

    Chen, Cao; Lv, Yan; Zhang, Bao-Yun; Zhang, Jin; Shi, Qi; Wang, Jing; Tian, Chan; Gao, Chen; Xiao, Kang; Ren, Ke; Zhou, Wei; Dong, Xiao-Ping


    It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP(C)) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP(C). Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP(C). Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.

  6. Generation of Reactive Oxygen Species (ROS) and Pro-Inflammatory Signaling in Human Brain Cells in Primary Culture. (United States)

    Lukiw, Walter J; Bjattacharjee, Surjyadipta; Zhao, Yuhai; Pogue, Aileen I; Percy, Maire E


    The cellular generation of reactive oxygen species (ROS) has been implicated in contributing to the pathology of human neurological disorders including Alzheimer's disease (AD) and Parkinson's disease (PD). To further understand the triggering and participation of ROS-generating species to pro-inflammatory and pathological signaling in human brain cells, in these experiments we studied the effects of 22 different substances (including various common drugs, interleukins, amyloid precursor protein, amyloid peptides and trace metals) at nanomolar concentrations, in a highly sensitive human neuronal-glial (HNG) cell primary co-culture assay. The evolution of ROS was assayed using the cell-permeate fluorescent indicator 2',7'-dichlorofluorescein diacetate (H2DCFDA), that reacts with major ROS species, including singlet oxygen, hydroxyl radicals or superoxides (λEx 488 nm; λEm 530 nm). Western analysis was performed for cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and cytosolic phospholipase A (cPLA2) to study the effects of induced ROS on inflammatory gene expression within the same brain cell sample. The data indicate that apart from acetylsalicylic acid (aspirin) and simvastatin, several neurophysiologically-relevant concentrations of Aβpeptides and neurotoxic trace metals variably induced ROS induction, COX-2 and cPLA2 expression. These findings have mechanistic implications for ROS-triggered inflammatory gene expression programs that may contribute to AD and PD neuropathologic mechanisms.

  7. Expression of miR-15/107 family microRNAs in human tissues and cultured rat brain cells. (United States)

    Wang, Wang-Xia; Danaher, Robert J; Miller, Craig S; Berger, Joseph R; Nubia, Vega G; Wilfred, Bernard S; Neltner, Janna H; Norris, Christopher M; Nelson, Peter T


    The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs), sharing a 5' AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively). In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS). In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs). Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  8. Effect of baicalin and berberine on transport of nimodipine on primary-cultured, rat brain microvascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Dong-mei ZHANG; Hai-yan LIU; Lin XIE; Xiao-dong LIU


    Aim: To investigate whether baicalin and berberine affects the transport of nimodipine (NMD) across the blood-brain barrier (BBB). Methods: Primary-cultured, rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the BBB. When cells became confluent, the steady-state uptake of NMD by rBMEC with or without baicalin and berberine was measured. The ef-fects of baicalin and berberine on the efflux of NMD from rBMEC were also studied.Results: Baicalin (2-5 μg/mL) increased the uptake of NMD, and baicalin (10-20 μg/mL) decreased the uptake. The steady-state uptake of NMD was higher than that of control group in the presence of 0.01-1 μg/mL berberine, but was lower in the presence of 2-10 μg/mL berberine. Conclusion: The bidirectional effect of baicalin and berberine on the uptake of NMD by rBMEC was found. Higher concentration showed an inhibitory effect, and lower concentration demonstrated an increasing effect.

  9. Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity: an animal-free CNS cell culture model. (United States)

    Woehrling, Elizabeth K; Hill, Eric J; Nagel, David; Coleman, Michael D


    Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

  10. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures. (United States)

    Pringle, A K; Sundstrom, L E; Wilde, G J; Williams, L R; Iannotti, F


    We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

  11. Anion-selective channelrhodopsin expressed in neuronal cell culture and in vivo in murine brain: Light-induced inhibition of generation of action potentials. (United States)

    Dolgikh, D A; Malyshev, A Yu; Salozhin, S V; Nekrasova, O V; Petrovskaya, L E; Roshchin, M V; Borodinova, A A; Feldman, T B; Balaban, P M; Kirpichnikov, M P; Ostrovsky, M A


    Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

  12. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp;

    Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming ...

  13. Secretion of nerve growth factor, brain-derived neurotrophic factor, and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

    Institute of Scientific and Technical Information of China (English)

    Sanjiang Feng; Minghua Zhuang; Rui Wu


    The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7–10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion. Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

  14. Effects of Carbon Ions on Primary Cultures of Mouse Brain Cells (United States)

    Nojima, K.; Ando, K.; Fujiwara, H.; Ando, S.

    Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/μm carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/μun carbon ions than 70 keV/μm carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality

  15. 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;


    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...

  16. Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Aβ and insulin degradation

    Directory of Open Access Journals (Sweden)

    Castaño Eduardo M


    Full Text Available Abstract Background Insulin degrading enzyme (IDE is implicated in the regulation of amyloid β (Aβ steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD. Although IDE sub-cellular localization has been well studied, the compartments relevant to Aβ degradation remain to be determined. Results Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Aβ and its distribution (DRMs vs. non-DRMs and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-β-cyclodextrin (MβCD, endogenous Aβ accumulated in the extracellular space and exogenous Aβ proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Aβ degradation in vitro. Conclusion Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Aβ accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for

  17. Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain. (United States)

    Gauthier, Sebastien A; Sahoo, Susmita; Jung, Sonia S; Levy, Efrat


    The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.

  18. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells. (United States)

    Tripathi, Vinay K; Kumar, Vivek; Singh, Abhishek K; Kashyap, Mahendra P; Jahan, Sadaf; Pandey, Ankita; Alam, Sarfaraz; Khan, Feroz; Khanna, Vinay K; Yadav, Sanjay; Lohani, Mohtshim; Pant, Aditya B


    The expression and metabolic profile of cytochrome P450s (CYPs) is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y) and glial (U373-MG) cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC), cyclophosphamide (CPA), ethanol and known neurotoxicant- monocrotophos (MCP), a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h) of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against xenobiotics.

  19. Monocrotophos induces the expression and activity of xenobiotic metabolizing enzymes in pre-sensitized cultured human brain cells.

    Directory of Open Access Journals (Sweden)

    Vinay K Tripathi

    Full Text Available The expression and metabolic profile of cytochrome P450s (CYPs is largely missing in human brain due to non-availability of brain tissue. We attempted to address the issue by using human brain neuronal (SH-SY5Y and glial (U373-MG cells. The expression and activity of CYP1A1, 2B6 and 2E1 were carried out in the cells exposed to CYP inducers viz., 3-methylcholanthrene (3-MC, cyclophosphamide (CPA, ethanol and known neurotoxicant- monocrotophos (MCP, a widely used organophosphorous pesticide. Both the cells show significant induction in the expression and CYP-specific activity against classical inducers and MCP. The induction level of CYPs was comparatively lower in MCP exposed cells than cells exposed to classical inducers. Pre-exposure (12 h of cells to classical inducers significantly added the MCP induced CYPs expression and activity. The findings were concurrent with protein ligand docking studies, which show a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR, PXR and AHR. Similarly, the known CYP inducers- 3-MC, CPA and ethanol have also shown significantly high docking scores with all the three studied CYP regulators. The expression of CYPs in neuronal and glial cells has suggested their possible association with the endogenous physiology of the brain. The findings also suggest the xenobiotic metabolizing capabilities of these cells against MCP, if received a pre-sensitization to trigger the xenobiotic metabolizing machinery. MCP induced CYP-specific activity in neuronal cells could help in explaining its effect on neurotransmission, as these CYPs are known to involve in the synthesis/transport of the neurotransmitters. The induction of CYPs in glial cells is also of significance as these cells are thought to be involved in protecting the neurons from environmental insults and safeguard them from toxicity. The data provide better understanding of the metabolizing capability of the human brain cells against

  20. Identification and culture of neural stem cells isolated from adult rat subventricular zone following fluid percussion brain injury

    Institute of Scientific and Technical Information of China (English)


    Objective To analyze proliferation and differentiation of glial fibrillary acid protein(GFAP)-and nestin-positive(GFAP+/nestin+)cells isolated from the subventricular zone following fluid percussion brain injury to determine whether GFAP+/nestin+ cells exhibit characteristics of neural stem cells.Methods Male Sprague-Dawley rats,aged 12 weeks and weighing 200-250 g,were randomly and evenly assigned to normal control group and model group.In the model group,a rat model of fluid percussion brain injury was es...

  1. Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture (United States)

    Maj, Magdalena; Milenkovic, Ivan; Bauer, Jan; Berggård, Tord; Veit, Martina; Ilhan-Mutlu, Aysegül; Wagner, Ludwig; Tretter, Verena


    Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer’s disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca2+. Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons. PMID:22888312

  2. Study of effect of oxygen/glucose-deprived culture on the brain-pancreas relative protein in PC12 cells and the mechanism

    Institute of Scientific and Technical Information of China (English)

    Yan-huaLIN; LuTIE; Ai-huaLIU; Xue-junLI


    AIM: To study the effect of oxygen/glucose-deprived (OGD)culture on the expression of a novel protein, brain-pancreas relative protein (BPRP), and the possible regulating mechanism in vitro. BPRP was a key protein found in our previous study of cerebral ischemia. METHODS: PC12 cells was selected and exposed to the Eagle's solution containing 1 mmol/L Na2S2O4 for

  3. Organotypic slice culture of embryonic brain tissue. (United States)

    Daza, Ray A M; Englund, Chris; Hevner, Robert F


    INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.

  4. Neuroglial cells in long-term primary cultures from the gilthead sea bream (Sparus aurata L.: new functional in vitro model from bony fish brain

    Directory of Open Access Journals (Sweden)

    Gerardo Centoducati


    Full Text Available Neuroglia has been historically considered the “glue” of the nervous system, as the ancient Greek name suggests, being simply referred as non-neuronal cells, with supporting functions for neurons in the CNS of mammalian and lower vertebrates. All around the world, approximately 283 cell lines were obtained from fish, yet none of these was from the brain of Sparus aurata, neither in cell lines nor as primary culture. Here we describe a novel in vitro reproducible neuroglial marine model for establishing primary neuroglial cell cultures, by dissociating the whole brain of seabream juveniles. We showed that proliferating neural stem cells produced alongside three generating lineages, such as neuronal precursor cells, astroglial precursor cells and oligodendroglia precursor cells, which developed respectively neurons, astrocytes and oligodendrocytes. The radial glia, finely described by morphological studies and immunochemical antigen expression, showed a peculiar spatial distribution, giving rise simultaneously both to astrocytes and neuronal precursors within a highly proliferative assemblate. Radial glia cells were assessed by glial fibrillary acidic protein (GFAP and vimentin reactivity, astrocytes by GFAP, neurons by the neuron-specific markers for ubiquitin carboxy-terminal hydrolase 1 (UCHL1 and intermediate filament associated protein (NF, whereas myelinating oligodendrocytes were immunostained with anti-myelin basic protein (MBP and anti-O4. Our findings suggest that seabream neuroglial cells gain in 3-4 weeks of culturing proliferation, neuroglial differentiation, and oligodendrocyte maturation with myelination, thus disclosing on the possibility that mixed neuroglial cultures can accelerate the maturation of oligodendrocytes and the regeneration of CNS injury in fish.

  5. Transport of monocarboxylic acids at the blood-brain barrier: Studies with monolayers of primary cultured bovine brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Terasaki, T.; Takakuwa, S.; Moritani, S.; Tsuji, A. (Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University (Japan))


    The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 {plus minus} 1.87 mM, the maximum uptake rate, Jmax, was 144.7 {plus minus} 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 {plus minus} 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of (3H)acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for (3H)acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of (3H)acetic acid at medium pH of 5.0 and 6.0, whereas 4,4{prime}-diisothiocyanostilben-2,2{prime}-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of (3H)acetic acid, whereas di- and tricarboxylic acids did not. The uptake of (3H)acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of (3H)acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH.

  6. Using Drawings of the Brain Cell to Exhibit Expertise in Neuroscience: Exploring the Boundaries of Experimental Culture (United States)

    Hay, David B.; Williams, Darren; Stahl, Daniel; Wingate, Richard J.


    This paper explores the research perspective of neuroscience by documenting the brain cell (neuron) drawings of undergraduates, trainee scientists, and leading neuroscience researchers in a single research-intensive university. Qualitative analysis, drawing-sorting exercises, and hierarchical cluster analysis are used to answer two related…

  7. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.


    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidoptera

  8. [Pharmacological assessment of ARTCEREB irrigation and perfusion solution for cerebrospinal surgery based on glucose incorporation activity in primary cultures of rat brain cells]. (United States)

    Nishimura, Masuhiro; Doi, Kazuhisa; Naito, Shinsaku


    ARTCEREB irrigation and perfusion solution (Artcereb) is typically used as an artificial fluid for applications inside the skull and spinal cavity. This in vitro study was conducted to assess the effects of Artcereb in cultures of rat fetal brain cells. Cell function following exposure to Artcereb was evaluated by measuring (3)H-deoxy-D-glucose incorporation activity. Cell function was significantly reduced in primary cultures of rat fetal brain cells at 0 h and 24 h after 1-h or 3-h exposure to normal saline as compared with Artcereb. Cell function was also significantly reduced at 24 h after 3-h exposure to lactated Ringer's solution as compared with Artcereb. Furthermore, cell function was significantly reduced at 24 h after 3-h exposure to a modified Artcereb formulation lacking either HCO(3)(-) or Mg(2+) as compared with Artcereb, while cell function was unaffected at 24 h after exposure to lactated Ringer's solution with HCO(3)(-) or normal saline with HCO(3)(-) as compared with Artcereb. These findings suggest the importance of the presence of HCO(3)(-) and Mg(2+) in the formulation of Artcereb.

  9. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain. (United States)

    Schou-Pedersen, Anne Marie V; Hansen, Stine N; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens


    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical detection provided limits of quantifications (LOQs) between 3.6 and 12nM. Within the linear range, obtained recoveries were from 90.9±9.9 to 120±14% and intra-day and inter-day precisions found to be less than 5.5% and 12%, respectively. The analytical method was applicable for quantification of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7pmol per 2 million cells intracellularly, but only the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid in frontal cortex, as compared to cerebellum. The chemical turnover in frontal cortex tissue of guinea pig was for serotonin successfully predicted from the turnover observed in the frontal cortex cell culture. In conclusion, the present analytical method shows high precision, accuracy and sensitivity and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry.

  10. Primary cortical brain cells influence osteoblast activity. (United States)

    Anissian, Lucas; Kirby, Michael; Stark, André


    The presence of neuropeptides and neuroreceptors in the bone have been reported in several studies. Bone turn-over seems to be controlled by the nervous system. The actual pathway or the control mechanism is still under investigation. In this study we investigate the changes in osteoblast cells if they are in co-culture with primary cortical brain cells. After seven days in co-culture with the primary fetal brain cells the osteoblast cells exhibited hypertrophic morphological changes and showed stronger ALP activity.

  11. Cell Culture Made Easy. (United States)

    Dye, Frank J.


    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…


    Directory of Open Access Journals (Sweden)

    PIZA Adriana Souza de Toledo


    Full Text Available An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera. In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.

  13. Molluscan cells in culture: primary cell cultures and cell lines



    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  14. A LED-based method for monitoring NAD(P)H and FAD fluorescence in cell cultures and brain slices. (United States)

    Rösner, Jörg; Liotta, Agustin; Schmitz, Dietmar; Heinemann, Uwe; Kovács, Richard


    Nicotinamide- and flavine-adenine-dinucleotides (NAD(P)H and FADH₂) are electron carriers involved in cellular energy metabolism and in a multitude of enzymatic processes. As reduced NAD(P)H and oxidised FAD molecules are fluorescent, changes in tissue auto-fluorescence provide valuable information on the cellular redox state and energy metabolism. Since fluorescence excitation, by mercury arc lamps (HBO) is inherently coupled to photo-bleaching and photo-toxicity, microfluorimetric monitoring of energy metabolism might benefit from the replacement of HBO lamps by light emitting diodes (LEDs). Here we describe a LED-based custom-built setup for monitoring NAD(P)H and FAD fluorescence at the level of single cells (HEK293) and of brain slices. We compared NAD(P)H bleaching characteristics with two light sources (HBO lamp and LED) as well as sensitivity and signal to noise ratio of three different detector types (multi-pixel photon counter (MPPC), photomultiplier tube (PMT) and photodiode). LED excitation resulted in reduced photo-bleaching at the same fluorescence output in comparison to excitation with the HBO lamp. Transiently increasing LED power resulted in reversible bleaching of NAD(P)H fluorescence. Recovery kinetics were dependent on metabolic substrates indicating coupling of NAD(P)H fluorescence to metabolism. Electrical stimulation of brain slices induced biphasic redox changes, as indicated by NAD(P)H/FAD fluorescence transients. Increasing the gain of PMT and decreasing the LED power resulted in similar sensitivity as obtained with the MPPC and the photodiode, without worsening the signal to noise ratio. In conclusion, replacement of HBO lamp with LED might improve conventional PMT based microfluorimetry of tissue auto-fluorescence.

  15. Culture: by the brain and in the brain? (United States)

    Ortega, Francisco; Vidal, Fernando


    Since the 1990s, several disciplines have emerged at the interface between neuroscience and the social and human sciences. For the most part, they aim at capturing the commonalities that underlay the heterogeneity of human behaviors and experiences. Neuroanthropology and cultural neuroscience, or the "neurodisciplines of culture," appear different, since their goal is to understand specificity rather than commonality and to address how cultural differences are inscribed in the brain. After offering an overview of these disciplines, and of their relation to endeavors such as cultural psychology and social neuroscience, this article discusses some of the most representative studies in the area in order to explore in which ways they are relevant for an understanding of culture.

  16. Long-term brain slice culturing in a microfluidic platform

    DEFF Research Database (Denmark)

    Vedarethinam, Indumathi; Avaliani, N.; Tønnesen, J.;


    In this work, we present the development of a transparent poly(methyl methacrylate) (PMMA) based microfluidic culture system for handling long-term brain slice cultures independent of an incubator. The different stages of system development have been validated by culturing GFP producing brain...... brain slice culturing for 16 days....

  17. Mammalian Cell Culture Simplified. (United States)

    Moss, Robert; Solomon, Sondra


    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  18. Fish stem cell cultures. (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan


    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  19. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong


    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  20. The blood-brain barrier in vitro using primary culture

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart

    of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part....... In the second part of the thesis, the ability of turning BCECs into protein factories is investigated using a non-viral gene carrier. Transfection and protein synthesis of BCECs cultured with confined BBB properties were found to be feasible without disrupting the BBB properties, although it was not possible...

  1. Neural differentiation of brain-derived neurotrophic factor-expressing human umbilical cord blood-derived mesenchymal stem cells in culture via TrkB-mediated ERK and β-catenin phosphorylation and following transplantation into the developing brain. (United States)

    Lim, Jung Yeon; Park, Sang In; Kim, Seong Muk; Jun, Jin Ae; Oh, Ji Hyeon; Ryu, Chung Hun; Jeong, Chang Hyun; Park, Sun Hwa; Park, Soon A; Oh, Wonil; Chang, Jong Wook; Jeun, Sin-Soo


    The ability of mesenchymal stem cells (MSCs) to differentiate into neural cells makes them potential replacement therapeutic candidates in neurological diseases. Presently, overexpression of brain-derived neurotrophic factor (BDNF), which is crucial in the regulation of neural progenitor cell differentiation and maturation during development, was sufficient to convert the mesodermal cell fate of human umbilical cord blood-derived MSCs (hUCB-MSCs) into a neuronal fate in culture, in the absence of specialized induction chemicals. BDNF overexpressing hUCB-MSCs (MSCs-BDNF) yielded an increased number of neuron-like cells and, surprisingly, increased the expression of neuronal phenotype markers in a time-dependent manner compared with control hUCB-MSCs. In addition, MSCs-BDNF exhibited a decreased labeling for MSCs-related antigens such as CD44, CD73, and CD90, and decreased potential to differentiate into mesodermal lineages. Phosphorylation of the receptor tyrosine kinase B (TrkB), which is a receptor of BDNF, was increased significantly in MSC-BDNF. BDNF overexpression also increased the phosphorylation of β-catenin and extracellular signal-regulated kinases (ERKs). Inhibition of TrkB availability by treatment with the TrkB-specific inhibitor K252a blocked the BDNF-stimulated phosphorylation of β-catenin and ERKs, indicating the involvement of both the β-catenin and ERKs signals in the BDNF-stimulated and TrkB-mediated neural differentiation of hUCB-MSCs. Reduction of β-catenin availability using small interfering RNA-mediated gene silencing inhibited ERKs phosphorylation. However, β-catenin activation was maintained. In addition, inhibition of β-catenin and ERKs expression levels abrogated the BDNF-stimulated upregulation of neuronal phenotype markers. Furthermore, MSC-BDNF survived and migrated more extensively when grafted into the lateral ventricles of neonatal mouse brain, and differentiated significantly into neurons in the olfactory bulb and

  2. Molluscan cells in culture: primary cell cultures and cell lines. (United States)

    Yoshino, T P; Bickham, U; Bayne, C J


    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  3. Culture of Mouse Neural Stem Cell Precursors


    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S


    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  4. Cell Culturing of Cytoskeleton (United States)


    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  5. Oscillating Cell Culture Bioreactor (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.


    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  6. A Culture-Behavior-Brain Loop Model of Human Development. (United States)

    Han, Shihui; Ma, Yina


    Increasing evidence suggests that cultural influences on brain activity are associated with multiple cognitive and affective processes. These findings prompt an integrative framework to account for dynamic interactions between culture, behavior, and the brain. We put forward a culture-behavior-brain (CBB) loop model of human development that proposes that culture shapes the brain by contextualizing behavior, and the brain fits and modifies culture via behavioral influences. Genes provide a fundamental basis for, and interact with, the CBB loop at both individual and population levels. The CBB loop model advances our understanding of the dynamic relationships between culture, behavior, and the brain, which are crucial for human phylogeny and ontogeny. Future brain changes due to cultural influences are discussed based on the CBB loop model.

  7. A brain slice culture model for studies of endogenous and exogenous precursor cell migration in the rostral migratory stream

    DEFF Research Database (Denmark)

    Tanvig, Mette; Blaabjerg, Morten; Andersen, Rikke K


    The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb (OB) in rodents. This migration has been well studied in vivo, but an organotypic in vitro model would facilitate more experimental investigations. Here we introduce...

  8. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin


    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...... and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers....

  9. L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells. (United States)

    Audesirk, G; Audesirk, T; Ferguson, C; Lomme, M; Shugarts, D; Rosack, J; Caracciolo, P; Gisi, T; Nichols, P


    The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2

  10. Microfluidic Cell Culture Device (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)


    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  11. Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers

    DEFF Research Database (Denmark)

    Hersom, Maria; Helms, Hans Christian; Pretzer, Natasia;


    across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated...... in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe......]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin...

  12. Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

    Directory of Open Access Journals (Sweden)

    Andreas Androutsellis-Theotokis

    Full Text Available BACKGROUND: New mechanisms that regulate neural stem cell (NSC expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen. CONCLUSIONS/SIGNIFICANCE: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

  13. Rescue of Brain Function Using Tunneling Nanotubes Between Neural Stem Cells and Brain Microvascular Endothelial Cells. (United States)

    Wang, Xiaoqing; Yu, Xiaowen; Xie, Chong; Tan, Zijian; Tian, Qi; Zhu, Desheng; Liu, Mingyuan; Guan, Yangtai


    Evidence indicates that neural stem cells (NSCs) can ameliorate cerebral ischemia in animal models. In this study, we investigated the mechanism underlying one of the neuroprotective effects of NSCs: tunneling nanotube (TNT) formation. We addressed whether the control of cell-to-cell communication processes between NSCs and brain microvascular endothelial cells (BMECs) and, particularly, the control of TNT formation could influence the rescue function of stem cells. In an attempt to mimic the cellular microenvironment in vitro, a co-culture system consisting of terminally differentiated BMECs from mice in a distressed state and NSCs was constructed. Additionally, engraftment experiments with infarcted mouse brains revealed that control of TNT formation influenced the effects of stem cell transplantation in vivo. In conclusion, our findings provide the first evidence that TNTs exist between NSCs and BMECs and that regulation of TNT formation alters cell function.

  14. Cell culture's spider silk road. (United States)

    Perkel, Jeffrey


    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

  15. A network analysis of developing brain cultures (United States)

    Christopoulos, V. N.; Boeff, D. V.; Evans, C. D.; Crowe, D. A.; Amirikian, B.; Georgopoulos, A.; Georgopoulos, A. P.


    We recorded electrical activity from four developing embryonic brain cultures (4-40 days in vitro) using multielectrode arrays (MEAs) with 60 embedded electrodes. Data were filtered for local field potentials (LFPs) and downsampled to 1 ms to yield a matrix of time series consisting of 60 electrode × 60 000 time samples per electrode per day per MEA. Each electrode time series was rendered stationary and nonautocorrelated by applying an ARIMA (25, 1, 1) model and taking the residuals (i.e. innovations). Two kinds of analyses were then performed. First, a pairwise crosscorrelation (CC) analysis (±25 1 ms lags) revealed systematic changes in CC with lag, day in vitro (DIV), and inter-electrode distance. Specifically, (i) positive CCs were 1.76× more prevalent and 1.44× stronger (absolute value) than negative ones, and (ii) the strength of CC increased with DIV and decreased with lag and inter-electrode distance. Second, a network equilibrium analysis was based on the instantaneous (1 ms resolution) logratio of the number of electrodes that were above or below their mean, called simultaneous departure from equilibrium, SDE. This measure possesses a major computational advantage over the pairwise crosscorrelation approach because it is very simple and fast to calculate, an important factor for the analysis of large networks. The results obtained with SDE covaried highly with CC over DIV, which further validates the usefulness of this measure as a computationally effective tool for large scale network analysis.

  16. Culturing the adolescent brain: what can neuroscience learn from anthropology? (United States)

    Choudhury, Suparna


    Cultural neuroscience is set to flourish in the next few years. As the field develops, it is necessary to reflect on what is meant by 'culture' and how this can be translated for the laboratory context. This article uses the example of the adolescent brain to discuss three aspects of culture that may help us to shape and reframe questions, interpretations and applications in cultural neuroscience: cultural contingencies of categories, cultural differences in experience and cultural context of neuroscience research. The last few years have seen a sudden increase in the study of adolescence as a period of both structural and functional plasticity, with new brain-based explanations of teenage behaviour being taken up in education, policy and medicine. However, the concept of adolescence, as an object of behavioural science, took shape relatively recently, not much more than a hundred years ago and was shaped by a number of cultural and historical factors. Moreover, research in anthropology and cross-cultural psychology has shown that the experience of adolescence, as a period of the lifespan, is variable and contingent upon culture. The emerging field of cultural neuroscience has begun to tackle the question of cultural differences in social cognitive processing in adults. In this article, I explore what a cultural neuroscience can mean in the case of adolescence. I consider how to integrate perspectives from social neuroscience and anthropology to conceptualize, and to empirically study, adolescence as a culturally variable phenomenon, which, itself, has been culturally constructed.

  17. Endotoxin-induced lung alveolar cell injury causes brain cell damage (United States)

    Rodríguez-González, Raquel; Ramos-Nuez, Ángela; Martín-Barrasa, José Luis; López-Aguilar, Josefina; Baluja, Aurora; Álvarez, Julián; Rocco, Patricia RM; Pelosi, Paolo


    Sepsis is the most common cause of acute respiratory distress syndrome, a severe lung inflammatory disorder with an elevated morbidity and mortality. Sepsis and acute respiratory distress syndrome involve the release of inflammatory mediators to the systemic circulation, propagating the cellular and molecular response and affecting distal organs, including the brain. Since it has been reported that sepsis and acute respiratory distress syndrome contribute to brain dysfunction, we investigated the brain-lung crosstalk using a combined experimental in vitro airway epithelial and brain cell injury model. Conditioned medium collected from an in vitro lipopolysaccharide-induced airway epithelial cell injury model using human A549 alveolar cells was subsequently added at increasing concentrations (no conditioned, 2%, 5%, 10%, 15%, 25%, and 50%) to a rat mixed brain cell culture containing both astrocytes and neurons. Samples from culture media and cells from mixed brain cultures were collected before treatment, and at 6 and 24 h for analysis. Conditioned medium at 15% significantly increased apoptosis in brain cell cultures 24 h after treatment, whereas 25% and 50% significantly increased both necrosis and apoptosis. Levels of brain damage markers S100 calcium binding protein B and neuron-specific enolase, interleukin-6, macrophage inflammatory protein-2, as well as matrix metalloproteinase-9 increased significantly after treating brain cells with ≥2% conditioned medium. Our findings demonstrated that human epithelial pulmonary cells stimulated with bacterial lipopolysaccharide release inflammatory mediators that are able to induce a translational clinically relevant and harmful response in brain cells. These results support a brain-lung crosstalk during sepsis and sepsis-induced acute respiratory distress syndrome. PMID:25135986

  18. Lymphoreticular cells in human brain tumours and in normal brain.



    The present investigation, using various rosetting assays of cell suspensions prepared by mechanical disaggregation or collagenase digestion, demonstrated lymphoreticular cells in human normal brain (cerebral cortex and cerebellum) and in malignant brain tumours. The study revealed T and B lymphocytes and their subsets (bearing receptors for Fc(IgG) and C3) in 5/14 glioma suspensions, comprising less than 15% of the cell population. Between 20-60% of cells in tumour suspensions morphologicall...

  19. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))


    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  20. Cell culture purity issues and DFAT cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)


    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  1. Brain repair: cell therapy in stroke

    Directory of Open Access Journals (Sweden)

    Kalladka D


    Full Text Available Dheeraj Kalladka, Keith W Muir Institute of Neuroscience and Psychology, University of Glasgow, Southern General Hospital, Glasgow, United Kingdom Abstract: Stroke affects one in every six people worldwide, and is the leading cause of adult disability. Some spontaneous recovery is usual but of limited extent, and the mechanisms of late recovery are not completely understood. Endogenous neurogenesis in humans is thought to contribute to repair, but its extent is unknown. Exogenous cell therapy is promising as a means of augmenting brain repair, with evidence in animal stroke models of cell migration, survival, and differentiation, enhanced endogenous angiogenesis and neurogenesis, immunomodulation, and the secretion of trophic factors by stem cells from a variety of sources, but the potential mechanisms of action are incompletely understood. In the animal models of stroke, both mesenchymal stem cells (MSCs and neural stem cells (NSCs improve functional recovery, and MSCs reduce the infarct volume when administered acutely, but the heterogeneity in the choice of assessment scales, publication bias, and the possible confounding effects of immunosuppressants make the comparison of effects across cell types difficult. The use of adult-derived cells avoids the ethical issues around embryonic cells but may have more restricted differentiation potential. The use of autologous cells avoids rejection risk, but the sources are restricted, and culture expansion may be necessary, delaying treatment. Allogeneic cells offer controlled cell numbers and immediate availability, which may have advantages for acute treatment. Early clinical trials of both NSCs and MSCs are ongoing, and clinical safety data are emerging from limited numbers of selected patients. Ongoing research to identify prognostic imaging markers may help to improve patient selection, and the novel imaging techniques may identify biomarkers of recovery and the mechanism of action for cell

  2. Cell culture purity issues and DFAT cells. (United States)

    Wei, Shengjuan; Bergen, Werner G; Hausman, Gary J; Zan, Linsen; Dodson, Michael V


    Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  3. Cell Delivery System for Traumatic Brain Injury (United States)


    REPORT Cell Delivery System for Traumatic Brain Injury 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: We have met all of the milestones outlined in this...COVERED (From - To) 18-Sep-2006 Standard Form 298 (Rev 8/98) Prescribed by ANSI Std. Z39.18 - 17-Mar-2008 Cell Delivery System for Traumatic Brain Injury Report...Manassero*, Justin Kim*, Maureen St Georges*, Nicole Esclamado* and Elizabeth Orwin. “Development of a Cell Delivery System for Traumatic Brain Injury Using

  4. Training stem cells for treatment of malignant brain tumors

    Institute of Scientific and Technical Information of China (English)

    Shengwen; Calvin; Li; Mustafa; H; Kabeer; Long; T; Vu; Vic; Keschrumrus; Hong; Zhen; Yin; Brent; A; Dethlefs; Jiang; F; Zhong; John; H; Weiss; William; G; Loudon


    The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for pa-tients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution(i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system.

  5. Cell culture compositions (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian


    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  6. Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression. (United States)

    Behnan, Jinan; Isakson, Pauline; Joel, Mrinal; Cilio, Corrado; Langmoen, Iver A; Vik-Mo, Einar O; Badn, Wiaam


    The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.

  7. Xanthine oxidase activity regulates human embryonic brain cells growth

    Directory of Open Access Journals (Sweden)

    Kevorkian G. A.


    Full Text Available Aim. Involvement of Xanthine Oxidase (XO; EC1.1.3.22 in cellular proliferation and differentiation has been suggested by the numerous investigations. We have proposed that XO might have undoubtedly important role during the development, maturation as well as the death of human embryos brain cells. Methods. Human abortion material was utilized for the cultivation of brain cells (E90. XO activity was measured by the formation of uric acid in tissue. Cell death was detected by the utility of Trypan Blue dye. Results. Allopurinol suppressed the XO activity in the brain tissue (0.12 ± 0.02; 0.20 ± 0.03 resp., p < 0.05. On day 12th the number of cells in the culture treated with the Allopurinol at the early stage of development was higher in comparison with the Control (2350.1 ± 199.0 vs 2123 ± 96 and higher in comparison with the late period of treatment (1479.6 ± 103.8, p < < 0.05. In all groups, the number of the dead cells was less than in Control, indicating the protective nature of Allopurinol as an inhibitor of XO. Conclusions. Allopurinol initiates cells proliferation in case of the early treatment of the human brain derived cell culture whereas at the late stages it has an opposite effect.

  8. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ailar Nakhlband


    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  9. Co-culture of astrocytes with neurons from injured brain A time-dependent dichotomy

    Institute of Scientific and Technical Information of China (English)

    Xiaojing Xu; Min Wang; Jing Liu; Jingya Lv; Yanan Hu; Huanxiang Zhang


    As supportive cells for neuronal growth and development, much effort has been devoted to the role of astrocytes in the normal state. However, the effect of the astrocytes after injury remains elusive. In the present study, neurons isolated from the subventricular zone of injured neonatal rat brains were co-cultured with astrocytes. After 6 days, these astrocytes showed a mature neuron-like appearance and the number of survivingneurons, primary dendrites and total branches was significantly higher than those at 3 days. The neurons began to shrink at 9 days after co-culture with shorter and thinner processes and the number of primary dendrites and total branches was significantly reduced. These experimental findings indicate that astrocytes in the injured brain promote the development of neurons in the early stages of co-culture while these cells reversely inhibit neuronal growth and development at the later states.

  10. Insect Cell Culture and Biotechnology

    Institute of Scientific and Technical Information of China (English)

    Robert R.Granados; Guoxun Li; G.W.Blissard


    The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.

  11. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B


    -induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use...... for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection....

  12. Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells. (United States)

    Kornblum, H I; Yanni, D S; Easterday, M C; Seroogy, K B


    The epidermal growth factor receptor family consists of four related tyrosine kinases: the epidermal growth factor receptor (EGF-R or ErbB), ErbB2, ErbB3, and ErbB4. These receptors are capable of extensive cross-activation upon the binding of their ligands - the EGF family of peptides for EGF-R and the neuregulins for ErbB3 and ErbB4. Since EGF-R is expressed by proliferating cells in the central nervous system (CNS), including multipotent CNS stem cells, we examined the expression of ErbB2, ErbB3 and ErbB4 in the germinal epithelia of the developing rat brain using in situ hybridization. ErbB2 and ErbB4 mRNAs were widely distributed within the germinal zones as early as E12. However, as development proceeded, ErbB2 mRNA was mainly present within the layers of cells immediately adjacent to the ventricular surface - the ventricular zone, while ErbB4 mRNA was predominantly expressed by subventricular zone cells, in the regions where these specialized germinal epithelia were present. ErbB3 mRNA distribution within germinal epithelia was more restricted, primarily confined to the diencephalon and rostral midbrain. Cultured neurospheres, which contain CNS stem cells, expressed ErbB2, ErbB4 and, to a lesser extent, ErbB3 protein as demonstrated by Western blot analysis. This expression declined during following differentiation. Heregulin-beta1, a neuregulin, had no effect on the proliferative capacity of neurospheres. Overall, our results indicate that ErbB2, ErbB3 and ErbB4 may play important and distinct roles in the genesis of the CNS. However, our in vitro data do not support a role for neuregulins in proliferation, per se, of CNS stem cells.

  13. Islam, brain death, and transplantation: culture, faith, and jurisprudence. (United States)

    Arbour, Richard; AlGhamdi, Hanan Mesfer Saad; Peters, Linda


    A significant gap exists between availability of organs for transplant and patients with end-stage organ failure for whom organ transplantation is the last treatment option. Reasons for this mismatch include inadequate approach to potential donor families and donor loss as a result of refractory cardiopulmonary instability during and after brainstem herniation. Other reasons include inadequate cultural competence and sensitivity when communicating with potential donor families. Clinicians may not have an understanding of the cultural and religious perspectives of Muslim families of critically ill patients who may be approached about brain death and organ donation. This review analyzes Islamic cultural and religious perspectives on organ donation, transplantation, and brain death, including faith-based directives from Islamic religious authorities, definitions of death in Islam, and communication strategies when discussing brain death and organ donation with Muslim families. Optimal family care and communication are highlighted using case studies and backgrounds illustrating barriers and approaches with Muslim families in the United States and in the Kingdom of Saudi Arabia that can improve cultural competence and family care as well as increase organ availability within the Muslim population and beyond.

  14. Depression after traumatic brain injury: a biopsychosocial cultural perspective. (United States)

    Roy, Durga; Jayaram, Geetha; Vassila, Alex; Keach, Shari; Rao, Vani


    There are several challenges in diagnosing and treating mental illness amongst South Asians. Often times, formulating a patient's case presentation cannot adequately be accomplished strictly using a biopsychosocial model. The cultural components play an imperative role in explaining certain psychiatric symptoms and can guide treatment. With the growing population of immigrants coming to the United States, many of which require treatment for mental illness, it is essential that clinicians be cognizant in incorporating cultural perspectives when treating such patients. The authors describe the case of a 24-year old South Asian male who suffered an exacerbation of a depressive syndrome after a traumatic brain injury. Using a biopsychosocial cultural approach, this case highlights how South Asian cultural values can contribute to and incite psychiatric symptoms while simultaneously providing protective drivers for treatment outcomes.

  15. Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

    Institute of Scientific and Technical Information of China (English)

    Guanqun Qiao; Qingquan Li; Gang Peng; Jun Ma; Hongwei Fan; Yingbin Li


    Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are stil unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc+/SV40Tag+/Tet-on+) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cel s and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cel s were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibril ary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibril ary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibril ary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cel s. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

  16. Stem cells to regenerate the newborn brain

    NARCIS (Netherlands)

    van Velthoven, C.T.J.


    Perinatal hypoxia-ischemia (HI) is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. In this thesis we investigate whether mesenchymal stem cells (MSC) regenerate the neonatal brain after HI injury. We show that transplantation of MSC after neonatal brain injury

  17. 9 CFR 101.6 - Cell cultures. (United States)


    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue...

  18. Transfer of opiorphin through a blood-brain barrier culture model. (United States)

    Bocsik, Alexandra; Darula, Zsuzsanna; Tóth, Géza; Deli, Mária A; Wollemann, Mária


    Opioid peptides are potent analgesics with therapeutic potential in the treatment of acute and chronic pain. Their efficacy is limited by peptidases (enkephalinases). Opiorphin pentapeptide (QRFSR) is the first characterized human endogenous inhibitor of enkephalinases. The peptide is able to increase the binding and affinity of endogenous opiates to mu opioid receptors; thus, the mechanism of opiorphin may provide a new therapeutic approach in pain management. The analgesic effect of opiorphin was proven in several earlier published in vitro and in vivo studies. Our aim was to test the transfer of opiorphin through a blood-brain barrier model for the first time. The flux of opiorphin was tested on a blood-brain barrier culture model consisting of rat brain endothelial, glial and pericyte cells. Brain endothelial cells in this triple co-culture model form tight monolayers characterized by transendothelial electrical resistance measurement. Relative quantity of the peptide was estimated by mass spectrometry. The transfer of opiorphin through the blood-brain barrier model was estimated to be ∼3%, whereas the permeability coefficient was 0.53 ± 1.36 × 10(-6) cm/s (n = 4). We also observed rapid conversion of N-terminal glutamine into pyroglutamic acid during the transfer experiments. Our results indicate that opiorphin crosses cultured brain endothelial cells in the absence of serum factors in a significant amount. This is in agreement with previous in vivo data showing potentiation of enkephalin-mediated antinociception. We suggest that opiorphin may have a potential as a centrally acting novel drug to treat pain.

  19. Cerebral and brain stem Langerhans cell histiocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Breidahl, W.H. (Dept. of Radiology, Royal Perth Hospital, Nedlands (Australia)); Ives, F.J. (Dept. of Radiology, Royal Perth Hospital, Nedlands (Australia)); Khangure, M.S. (Dept. of Magnetic Resonance Imaging, Sir Charles Gairdner Hospital, Nedlands (Australia))


    Two patients with central nervous system manifestations of Langerhans cell histiocytosis, both with brain stem involvement, are reported. The onset of symptoms was at an age when the diagnosis might not have been considered. (orig.)

  20. Defining viability in mammalian cell cultures


    Browne, Susan M.; Al-Rubeai, Mohamed


    Abstract A large number of assays are available to monitor viability in mammalian cell cultures with most defining loss of viability as a loss of plasma membrane integrity, a characteristic of necrotic cell death. However, the majority of cultured cells die by apoptosis and early apoptotic cells, although non-viable, maintain an intact plasma membrane and are thus ignored. Here we measure the viability of cultures of a number of common mammalian cell lines by assays that measure me...

  1. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila


    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  2. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans


    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  3. Permeability of PEGylated Immunoarsonoliposomes Through In Vitro Blood Brain Barrier-Medulloblastoma Co-culture Models for Brain Tumor Therapy

    NARCIS (Netherlands)

    Al-Shehri, A.; Favretto, M.E.; Ioannou, P.V.; Romero, I.A.; Couraud, P.O.; Weksler, B.B.; Parker, T.L.; Kallinteri, P.


    PURPOSE: 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 therapeuti

  4. [Rat brain cells containing ezrin (cytovillin)]. (United States)

    Korzhevskiĭ, D E; Kirik, O V; Giliarov, A V


    Ezrin (cytovillin or p81 protein) is an actin-binding protein, a member of ERM (ezrin, radixin and moesin) family, which species contribute to stabilization of the plasma membrane-formed structures. The aim of the present study was to demonstrate the ezrin-containing cells in the rat brain and to describe their topography and morphological features. The most pronounced immunohistochemical reaction to ezrin was found in the epithelium of the choroid plexus, cells of the subcommissural organ and ventricular ependyma. Moreover, ezrin staining was also detected in the unidentifiable cells in the subventricular zone, rostral migration pathway and astrocytes in various brain areas. Preferential ezrin localization in the brain cells contributing to formation of barrier structures suggests its involvement in transport processes in the CNS.

  5. Fluidic system for long-term in vitro culturing and monitoring of organotypic brain slices

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Troels-Smith, Ane R.; Dimaki, Maria


    Brain slice preparations cultured in vitro have long been used as a simplified model for studying brain development, electrophysiology, neurodegeneration and neuroprotection. In this paper an open fluidic system developed for improved long term culturing of organotypic brain slices is presented. ...

  6. Mesenchymal stromal cells for traumatic brain injury




    The multiple pathological cascades activated after traumatic brain injury (TBI) and their extended nature offer the possibility for therapeutic interventions possibly affecting multiple injury mechanisms simultaneously. Mesenchymal stromal cell (MSC) therapy matches this need, being a bioreactor of a variety of molecules able to interact and modify the injured brain microenvironment. Compared to autologous MSCs, bank stored GMP-graded allogenic MSCs appear to be a realistic choice for TBI ...

  7. In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel

    NARCIS (Netherlands)

    Shi, Fuxin; Gounko, Natasha V.; Wang, Xiaoqin; Ronken, Eric; Hoekstra, Dick


    Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds c

  8. Cell density monitoring and control of microencapsulated CHO cell cultures


    Cole, Harriet Emma


    Though mammalian cells play a key role in the manufacturing of recombinant glycosylated proteins, cell cultures and productivity are limited by the lack of suitable systems to enable stable perfusion culture. Microencapsulation, or entrapping cells within a semi-permeable membrane, offers the potential to generate high cell density cultures and improve the productivity by mimicking the cells natural environment. However, the cells being secluded by the microcapsules membrane are difficult to ...

  9. Cell culture techniques in honey bee research (United States)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  10. Cell Culture as an Alternative in Education. (United States)

    Nardone, Roland M.


    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  11. Primary Culture of Porcine Pancreatic Acinar Cells



    OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3)H-thymidine incorporation of acinar cells and the activity of amylase or l...

  12. Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes%大鼠脑微血管内皮细胞与周细胞、星形胶质细胞共培养建立体外血脑屏障模型

    Institute of Scientific and Technical Information of China (English)

    查雨锋; 傅晓钟; 张顺; 罗敏; 欧瑜; 董永喜; 王爱民; 王永林


    目的:应用原代培养的大鼠脑微血管内皮细胞(brain-microvessel endothelial cells,BMECs )与脑微血管周细胞(brain-microvessel pericytes,BMPC )、星形胶质细胞(astro-cytes,AS)共培养建立可模拟在体状态的体外血脑屏障(blood-brain barrier,BBB)模型。方法原代分离、纯化和培养大鼠BMECs、BMPC和AS,通过细胞形态学和免疫细胞化学染色方法鉴定原代培养的细胞,应用Millicell细胞培养插(孔径0.4μm)建立5种不同类型的体外BBB模型,经跨内皮电阻值(transendothelial electrical resistance,TEER)、荧光素钠通透性(sodium fluorescent,Na-FLU )、碱性磷酸酶(AKP)和γ-谷氨酰转肽酶(γ-GT1)的表达测定以及阳性药在体内和体外BBB通透量的相似性,比较评价其屏障功能。结果原代培养的BMECs呈典型的铺路卵石样结构,BMPC胞体较大且呈分枝状,AS 有细长突触,胞质较浅;免疫细胞化学染色证实原代细胞为目标细胞;BMECs与BMPC、AS共培养后TEER值可达(478±25)Ω·cm2,Na-FLU 的表观渗透系数为[(8.23±0.78)×10-6]cm·s-1,AKP和γ-GT1表达分别为(6.90±0.27)金氏单位· g-1 Pro,(4.39±0.32)μg·g-1 Pro;阳性药在体外BBB的表观渗透系数(apparent permeability coefficient,Papp )与在体数据具有较好的相关性(R2=0.92)。结论原代培养的大鼠BMECs与BMPC、AS共培养建立的体外BBB模型在形态、结构及屏障功能方面具备BBB的基本特征,为研究BBB的生理学、病理学以及筛选化合物提供了一种有用工具。%Aim To establish in vitro blood-brain barrier (BBB) model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells (BMECs) with brain-microvessel pericytes (BMPC)and astrocytes (AS). Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then

  13. Functional bitter taste receptors are expressed in brain cells. (United States)

    Singh, Nisha; Vrontakis, Maria; Parkinson, Fiona; Chelikani, Prashen


    Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain.

  14. Culture of Cells from Amphibian Embryos. (United States)

    Stanisstreet, Martin


    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  15. Propagation of prions causing synucleinopathies in cultured cells. (United States)

    Woerman, Amanda L; Stöhr, Jan; Aoyagi, Atsushi; Rampersaud, Ryan; Krejciova, Zuzana; Watts, Joel C; Ohyama, Takao; Patel, Smita; Widjaja, Kartika; Oehler, Abby; Sanders, David W; Diamond, Marc I; Seeley, William W; Middleton, Lefkos T; Gentleman, Steve M; Mordes, Daniel A; Südhof, Thomas C; Giles, Kurt; Prusiner, Stanley B


    Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)-YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson's disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

  16. Primary Culture of Porcine Pancreatic Acinar Cells

    Directory of Open Access Journals (Sweden)

    Zhao X


    Full Text Available OBJECTIVE: To develop a method for the primary culture of porcine pancreatic acinar cells. INTERVENTIONS: Dispersed pancreatic acinar cells available utilizing RPMI-1640 medium containing collagenase III. After purification, the isolated acinar cells were cultured in RPMI-1640 medium with the addition of 2.5% fetal bovine serum. MAIN OUTCOME MEASURES: The morphological characteristics of acinar cells were described. (3H-thymidine incorporation of acinar cells and the activity of amylase or lipase were determined during the culture process. RESULTS: There were no remarkable morphological changes in the pancreatic acinar cells during the 20 days culture. The acini showed a tendency to gather but did not attach to the walls of the culture disks. A good (3H-thymidine incorporation of acinar cells in the primary culture was maintained. The secretion of amylase or lipase from the acini decreased with the length of time of the culture. DISCUSSION: The primary culture of acinar cells from a porcine pancreas which was carried out in this study maintained the normal morphology of the acinar cells and their ability to grow but not their secretion of amylase or lipase. The method would benefit by the further experiments on acini of porcine pancreas.

  17. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures. (United States)

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren


    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  18. Oscar Wilde and the brain cell. (United States)

    Cohn, Elisha


    This chapter considers Oscar Wilde's interest in the brain cell as an aesthetic object. Offering an account of Wilde's career that analyzes his early interest in physiology and philosophy, this chapter argues that Wilde's uniquely aesthetic take on the brain suggests that he rejects an account of the self as autonomous or self-determining. For many late Victorians brain science threatened both the freedom of human action and the legitimacy of beauty because it had the potential to invalidate conscious experience. But writers whose work Wilde knew, like John Ruskin, W. K. Clifford, and John Tyndall, avoided the despair of materialism by using aesthetic terms in their own discussions of life's invisible materials. Wilde's art collaborates with the contemporary sciences. His depictions of the cell direct the senses to a new field of being that emphasizes the molecular life all humans have in common, in which individual responsibility and activity matter less than the necessity of beauty.

  19. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)


    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  20. Embryonic Stem Cells: Isolation, Characterization and Culture (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  1. Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes

    DEFF Research Database (Denmark)

    Jahnsen, Henrik; Kristensen, Bjarne Winther; Thiébaud, P


    such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons......Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish...

  2. [Effects of beryllium chloride on cultured cells]. (United States)

    Sakaguchi, T; Sakaguchi, S; Nakamura, I; Kagami, M


    The effects of beryllium on cultured cells were investigated. Three cell-lines (HeLa-S3, Vero, HEL-R66) were used in these experiments and they were cultured in Eagle's MEM plus 5 or 10% FBS (Fetal Bovine Serum) containing beryllium in various concentrations. HeLa cells or Vero cells were able to grow in the medium with 10 micrograms Be/ml (1.1 mM). On the other hand, the growth of HEL cells were strongly inhibited, even when cultured in the medium with 1 microgram Be/ml (1.1 X 10(-1) mM) and the number of living cells showed markedly low level as compared to that of the control samples cultured in the medium without beryllium. The cytotoxic effects of beryllium on these cells, which were cultured for three days in the medium with beryllium, were observed. None of cytotoxic effects were found on HeLa cells cultured with 0.5 micrograms/ml (5.5 X 10(-2) mM) and on Vero cells cultured with 0.05 micrograms Be/ml (5.5 X 10(-3) mM), while HEL cells received cytotoxic effects even when cultured in the medium containing 0.05 micrograms Be/ml (5.5 X 10(-3) mM), and these effects on the cells appeared strong when cultured in the medium without FBS. It was revealed from these experiments that HEL cells are very sensitive in terms of toxic effects of beryllium. Therefore, there cells can be used for the toxicological study on low level concentrations of the metal.

  3. Brain Injury with Sickle Cell Disease

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap


    Full Text Available The relationship between brain injury and vasculopathy in 146 sickle cell (SCD patients with hemoglobin SS, the most serious form of SCD, was evaluated by MRI and MRA at St Jude Children’s Research Hospital, Memphis, TN.

  4. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair

    DEFF Research Database (Denmark)

    Noraberg, Jens; Poulsen, Frantz Rom; Blaabjerg, Morten


    ), Alzheimer's disease (AD) and epilepsia. Studies of non-excitotoxic neurotoxic compounds and the experimental use of slice cultures in studies of HIV neurotoxicity, traumatic brain injury (TBI) and neurogenesis are included. For cerebral ischemia, experimental models with oxygen-glucose deprivation (OGD......) and exposure to glutamate receptor agonists (excitotoxins) are reviewed. For epilepsia, focus is on induction of seizures with effects on neuronal loss, axonal sprouting and neurogenesis. For Alzheimer's disease, the review centers on the use of beta-amyloid (Abeta) in different models, while the section...... on repair is focused on neurogenesis and cell migration. The culturing techniques, set-up of models, and analytical tools, including markers for neurodegeneration, like the fluorescent dye propidium iodide (PI), are reviewed and discussed. Comparisons are made between hippocampal slice cultures and other...

  5. Uptake of SPECT radiopharmaceuticals in neocortical brain cultures

    Energy Technology Data Exchange (ETDEWEB)

    Jong, B.M. de; Royen, E.A. van


    The uptake, retention and uptake antagonism of /sup 201/Tl-DDC, /sup 201/Tl-Cl, /sup 123/I-IMP, /sup 99m/Tc-HMPAO and /sup 99m/Tc-O4/sup -/ were compared in rat neocortex cultures. /sup 201/Tl-DDC and /sup 123/I-IP revealed the highest uptake of radioactivity in the cultures. /sup 99m/Tc-HMPAO and /sup 123/I-IMP showed the highest retention of radioactivity within the tissue in washout experiments. Blocking of bioelectric activity by tetrodotoxin did not significantly affect the uptake of the radiopharmaceuticals (RPHA). Inhibition of Na K ATPase by ouabain inhibited the uptake of /sup 201/Tl-Cl (77%) and /sup 201/Tl-DDC (27%). Imipramine showed a significantly stronger inhibitory effect on /sup 123/I-IMP uptake in comparison with the effect on other RPHA. /sup 99m/Tc-O4/sup -/ was not concentrated within the cultured tissue. Under the in vitro conditions used in this study, the various RPHA were characterised by distinct differences in their interaction with cortical brain tissue.

  6. Anticancer and antioxidant properties of terpinolene in rat brain cells. (United States)

    Aydin, Elanur; Türkez, Hasan; Taşdemir, Sener


    Terpinolene (TPO) is a natural monoterpene present in essential oils of many aromatic plant species. Although various biological activities of TPO have been demonstrated, its neurotoxicity has never been explored. In this in vitro study we investigated TPO's antiproliferative and/or cytotoxic properties using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test, genotoxic damage potential using the single-cell gel electrophoresis (SCGE), and oxidative effects through total antioxidant capacity (TAC) and total oxidative stress (TOS) in cultured primary rat neurons and N2a neuroblastoma cells. Dose-dependent effects of TPO (at 10 mg L(-1), 25 mg L(-1), 50 mg L(-1), 100 mg L(-1), 200 mg L(-1), and 400 mg L(-1)) were tested in both cell types. Significant (P<0.05) decrease in cell proliferation were observed in cultured primary rat neurons starting with the dose of 100 mg L(-1) and in N2a neuroblastoma cells starting with 50 mg L(-1). TPO was not genotoxic in either cell type. In addition, TPO treatment at 10 mg L(-1), 25 mg L(-1), and 50 mg L(-1) increased TAC in primary rat neurons, but not in N2a cells. However, at concentrations above 50 mg L(-1) it increased TOS in both cell types. Our findings clearly demonstrate that TPO is a potent antiproliferative agent for brain tumour cells and may have potential as an anticancer agent, which needs to be further studied.

  7. Tropism mechanism of stem cells targeting injured brain tissues by stromal cell-derived factor-1

    Institute of Scientific and Technical Information of China (English)

    ZHANG Sai; LIU Xiao-zhi; LIU Zhen-lin; SHANG Chong-zhi; HU Qun-liang


    Objective: To explore the role and function of stromal cell-derived factor- 1 (SDF- 1) in stem cells migrating into injured brain area.Methods: Rat-derived nerve stem cells (NSCs) were isolated and cultured routinely. Transwell system was used to observe the migration ability of NSCs into injured nerve cells. Immunocytochemistry was used to explore the expression of chemotactic factor receptor-4 (CXCR-4) in NSCs. In vivo, we applied immunofluorescence technique to observe the migration of NSCs into injured brain area. Immunofluorescence technique and Western blotting were used to test expression level of SDF- 1. After AMD3100 (a special chemical blocker) blocking CXCR-4, the migration ability of NSCs was tested in vivo and in vitro, respectively.Results: NSCs displayed specific tropism for injured nerve cells or traumatic brain area in vivo and in vitro. The expression level of SDF-1 in traumatic brain area increased remarkably and the expression level of CXCR-4 in the NSCs increased simultaneously. After AMD3100 blocking the expression of CXCR-4, the migration ability of NSCs decreased significantly both in vivo and in vitro.Conclusions: SDF-1 may play a key role in stem cells migrating into injured brain area through specially combining with CXCR-4.

  8. Neocortical glial cell numbers in human brains

    DEFF Research Database (Denmark)

    Pelvig, D.P.; Pakkenberg, H.; Stark, A.K.;


    Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia...... while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males...

  9. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Audus, K.L.; Borchardt, R.T.


    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.

  10. Apoptotic markers in cultured fibroblasts correlate with brain metabolites and regional brain volume in antipsychotic-naive first-episode schizophrenia and healthy controls. (United States)

    Batalla, A; Bargalló, N; Gassó, P; Molina, O; Pareto, D; Mas, S; Roca, J M; Bernardo, M; Lafuente, A; Parellada, E


    Cultured fibroblasts from first-episode schizophrenia patients (FES) have shown increased susceptibility to apoptosis, which may be related to glutamate dysfunction and progressive neuroanatomical changes. Here we determine whether apoptotic markers obtained from cultured fibroblasts in FES and controls correlate with changes in brain glutamate and N-acetylaspartate (NAA) and regional brain volumes. Eleven antipsychotic-naive FES and seven age- and gender-matched controls underwent 3-Tesla magnetic resonance imaging scanning. Glutamate plus glutamine (Glx) and NAA levels were measured in the anterior cingulate (AC) and the left thalamus (LT). Hallmarks of apoptotic susceptibility (caspase-3-baseline activity, phosphatidylserine externalization and chromatin condensation) were measured in fibroblast cultures obtained from skin biopsies after inducing apoptosis with staurosporine (STS) at doses of 0.25 and 0.5 μM. Apoptotic biomarkers were correlated to brain metabolites and regional brain volume. FES and controls showed a negative correlation in the AC between Glx levels and percentages of cells with condensed chromatin (CC) after both apoptosis inductions (STS 0.5 μM: r = -0.90; P = 0.001; STS 0.25 μM: r = -0.73; P = 0.003), and between NAA and cells with CC (STS 0.5 μM induction r = -0.76; P = 0.002; STS 0.25 μM r = -0.62; P = 0.01). In addition, we found a negative correlation between percentages of cells with CC and regional brain volume in the right supratemporal cortex and post-central region (STS 0.25 and 0.5 μM; P < 0.05 family-wise error corrected (FWEc)). We reveal for the first time that peripheral markers of apoptotic susceptibility may correlate with brain metabolites, Glx and NAA, and regional brain volume in FES and controls, which is consistent with the neuroprogressive theories around the onset of the schizophrenia illness.

  11. 大鼠原代脑微血管内皮细胞体外分离与培养的实验研究%In Vitro Isolation and Culture of Primary Rat Brain Microvascular Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    李振; 刘云会; 薛一雪; 刘丽波; 王萍


    目的 探讨获取大鼠脑微血管内皮细胞(BMEC)的简单、有效方法,为构建体外血肿瘤屏障(BTB)模型提供材料.方法 采集出生3~5 d的Wistar胎鼠大脑皮质,应用酶消化法及葡聚糖离心法获得脑微血管段后,接种于培养皿中进行原代培养,采用倒置显微镜对所培养的细胞进行形态学观察;以Ⅷ因子相关抗原免疫组化染色法鉴定细胞;将BMEC与C6脑胶质瘤细胞共培养,构建体外BTB模型,并采用免疫组化法和免疫荧光法检测BMEC间紧密连接相关蛋白occludin的表达.结果 体外培养2h时脑微血管段贴壁,12~48 h见圆形生发中心形成,2~3d单层内皮细胞自生发中心长出,4~5 d见较大单层内皮细胞团,5~7 d可见融合成片的内皮细胞单层,外观呈“铺路石”样;第Ⅷ因子免疫组化结果显示“铺路石”样细胞胞质呈棕黄色染色;紧密连接相关蛋白occludin的免疫组化和荧光结果证明共培养的BMEC间表达BTB的特性.结论 本方法能成功地进行大鼠原代BMEC培养,构建大鼠体外BTB模型,进而应用于BTB的生理、生化及药理学研究.%Objective To explore a simple and reprodueable method lor the in vitro isolation and eulture of rat brain microvascular endothelial cells (BMEC), so as to provide materials for the construction of in vilro blood tumor barrier ( BTB) models. Methods Relative!* pure cerebral microvessel fragments were obtained from the cortex of 3-5 days old wistar rats through dissection,enzyme digestion,and dex-tran centrifugation. Then, these fragments were seeded on dishes for primary culture. The morphology of BMEC was observed under an invert microscope. BMEC were identified by immunohistoehemistry with factor VIII-associated antigen. In vitro BTB models were constructed by co-cultivation of BMEC with C6 glioma cells. The expression of tight junction-related protein oeeludin in BMEC of BTB was measured by immunohistoehemistry and

  12. Dynamic culture improves cell reprogramming efficiency. (United States)

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song


    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  13. Autofluorescence of viable cultured mammalian cells. (United States)

    Aubin, J E


    The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.

  14. Liposome-siRNA-peptide complexes cross the blood-brain barrier and significantly decrease PrP on neuronal cells and PrP in infected cell cultures.

    Directory of Open Access Journals (Sweden)

    Bruce Pulford

    Full Text Available BACKGROUND: Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP(C expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP(C expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs, acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation. CONCLUSIONS/SIGNIFICANCE: Liposome-siRNA-peptide complexes (LSPCs delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP(C expression and eliminated PrP(RES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP(C-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

  15. Emulsions Containing Perfluorocarbon Support Cell Cultures (United States)

    Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.


    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

  16. Cell culture processes for monoclonal antibody production


    LI Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert; Amanullah, Ashraf


    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizin...

  17. The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Geppert, Mark; Petters, Charlotte [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany); Thiel, Karsten [Fraunhofer Institute for Manufacturing Technology and Advanced Materials (Germany); Dringen, Ralf, E-mail: [University of Bremen, Centre for Biomolecular Interactions Bremen (Germany)


    Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood-brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 {+-} 2 to 107 {+-} 6 nm and the zeta-potential of the particles from -22 {+-} 5 to -9 {+-} 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

  18. Organotypic brain slice cultures of adult transgenic P301S mice--a model for tauopathy studies.

    Directory of Open Access Journals (Sweden)

    Agneta Mewes

    Full Text Available BACKGROUND: Organotypic brain slice cultures represent an excellent compromise between single cell cultures and complete animal studies, in this way replacing and reducing the number of animal experiments. Organotypic brain slices are widely applied to model neuronal development and regeneration as well as neuronal pathology concerning stroke, epilepsy and Alzheimer's disease (AD. AD is characterized by two protein alterations, namely tau hyperphosphorylation and excessive amyloid β deposition, both causing microglia and astrocyte activation. Deposits of hyperphosphorylated tau, called neurofibrillary tangles (NFTs, surrounded by activated glia are modeled in transgenic mice, e.g. the tauopathy model P301S. METHODOLOGY/PRINCIPAL FINDINGS: In this study we explore the benefits and limitations of organotypic brain slice cultures made of mature adult transgenic mice as a potential model system for the multifactorial phenotype of AD. First, neonatal (P1 and adult organotypic brain slice cultures from 7- to 10-month-old transgenic P301S mice have been compared with regard to vitality, which was monitored with the lactate dehydrogenase (LDH- and the MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assays over 15 days. Neonatal slices displayed a constant high vitality level, while the vitality of adult slice cultures decreased significantly upon cultivation. Various preparation and cultivation conditions were tested to augment the vitality of adult slices and improvements were achieved with a reduced slice thickness, a mild hypothermic cultivation temperature and a cultivation CO(2 concentration of 5%. Furthermore, we present a substantial immunohistochemical characterization analyzing the morphology of neurons, astrocytes and microglia in comparison to neonatal tissue. CONCLUSION/SIGNIFICANCE: Until now only adolescent animals with a maximum age of two months have been used to prepare organotypic brain slices. The current study

  19. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen


    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  20. Insect cell culture in reagent bottles. (United States)

    Rieffel, S; Roest, S; Klopp, J; Carnal, S; Marti, S; Gerhartz, B; Shrestha, B


    Growing insect cells with high air space in culture vessel is common from the early development of suspension cell culture. We believed and followed it with the hope that it allows sufficient air for optimal cell growth. However, we missed to identify how much air exactly cells need for its growth and multiplication. Here we present the innovative method that changed the way we run insect cell culture. The method is easy to adapt, cost-effective and useful for both academic and industrial research labs. We believe this method will revolutionize the way we run insect cell culture by increasing throughput in a cost-effective way. In our study we identified:•Insect cells need to be in suspension; air space in culture vessel and type of culture vessel is of less importance. Shaking condition that introduces small air bubbles and maintains it in suspension for longer time provides better oxygen transfer in liquid. For this, high-fill volume in combination with speed and shaking diameter are important.•Commercially available insect cells are not fragile as original isolates. These cells can easily withstand higher shaking speed.•Growth condition in particular lab set-up needs to be optimized. The condition used in one lab may not be optimum for another lab due to different incubators from different vendors.

  1. Human primary brain tumor cell growth inhibition in serum-free medium optimized for neuron survival. (United States)

    Brewer, Gregory J; LeRoux, Peter D


    Glioblastoma is the most common primary brain tumor in adults from which about 15,000 patients die each year in the United States. Despite aggressive surgery, radiotherapy and chemotherapy, median survival remains only 1 year. Here we evaluate growth of primary human brain tumor cells in a defined nutrient culture medium (Neuregen) that was optimized for neuron regeneration. We hypothesized that Neuregen would inhibit tumor cell growth because of its ability to inhibit gliosis in rat brain. Tumor tissue was collected from 18 patients including 10 males and 8 females (mean age 60+/-12 years) who underwent craniotomy for newly diagnosed, histologically confirmed brain tumors. The tissue was shipped overnight in Hibernate transport medium. Tumor cells were isolated and plated in Neurobasal/serum or Neuregen on culture plastic. After 1 week, growth in Neuregen was significantly less in 9/10 glioblastoma multiforme cases, 5/5 meningioma cases and 3/3 cases of brain metastasis. Analysis of deficient formulations of Neuregen and formulations to which selected components were added back implicate no single active component. However, individual cases were sensitive to corticosterone, selenium, ethanolamine, fatty acids and/or antioxidants. Therefore, a defined culture medium that promotes neuron regeneration inhibits the growth of human primary glioblastoma, meningioma and metastatic tumor cells in culture. The possible in vivo efficacy of Neuregen for treatment of brain tumor resections remains to be determined.

  2. Human microglial cells synthesize albumin in brain.

    Directory of Open Access Journals (Sweden)

    Sung-Min Ahn

    Full Text Available Albumin, an abundant plasma protein with multifunctional properties, is mainly synthesized in the liver. Albumin has been implicated in Alzheimer's disease (AD since it can bind to and transport amyloid beta (Abeta, the causative agent of AD; albumin is also a potent inhibitor of Abeta polymerization. Despite evidence of non-hepatic transcription of albumin in many tissues including kidney and pancreas, non-hepatic synthesis of albumin at the protein level has been rarely confirmed. In a pilot phase study of Human Brain Proteome Project, we found evidence that microglial cells in brain may synthesize albumin. Here we report, for the first time, the de novo synthesis of albumin in human microglial cells in brain. Furthermore, we demonstrate that the synthesis and secretion of albumin from microglial cells is enhanced upon microglial activation by Abeta(1-42- or lipopolysaccharide (LPS-treatment. These data indicate that microglial cells may play a beneficial role in AD by secreting albumin that not only inhibits Abeta polymerization but also increases its clearance.

  3. Hyperforin changes the zinc-storage capacities of brain cells. (United States)

    Gibon, Julien; Richaud, Pierre; Bouron, Alexandre


    In vitro and in vivo experiments were carried out to investigate the consequences on brain cells of a chronic treatment with hyperforin, a plant extract known to dissipate the mitochondrial membrane potential and to release Zn(2+) and Ca(2+) from these organelles. Dissociated cortical neurons were grown in a culture medium supplemented with 1 μM hyperforin. Live-cell imaging experiments with the fluorescent probes FluoZin-3 and Fluo-4 show that a 3 day-hyperforin treatment diminishes the size of the hyperforin-sensitive pools of Ca(2+) and Zn(2+) whereas it increases the size of the DTDP-sensitive pool of Zn(2+) without affecting the ionomycin-sensitive pool of Ca(2+). When assayed by quantitative PCR the levels of mRNA coding for metallothioneins (MTs) I, II and III were increased in cortical neurons after a 3 day-hyperforin treatment. This was prevented by the zinc chelator TPEN, indicating that the plant extract controls the expression of MTs in a zinc-dependent manner. Brains of adult mice who received a daily injection (i.p.) of hyperforin (4 mg/kg/day) for 4 weeks had a higher sulphur content than control animals. They also exhibited an enhanced expression of the genes coding for MTs. However, the long-term treatment did not affect the brain levels of calcium and zinc. Based on these results showing that hyperforin influences the size of the internal pools of Zn(2+), the expression of MTs and the brain cellular sulphur content, it is proposed that hyperforin changes the Zn-storage capacity of brain cells and interferes with their thiol status.

  4. Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting

    Institute of Scientific and Technical Information of China (English)

    Xiuping Zhou; Chao Zheng; Qiong Shi; Xiang Li; Zhigang Shen; Rutong Yu


    This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

  5. Rotating cell culture systems for human cell culture: human trophoblast cells as a model. (United States)

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin


    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS

  6. Porcine mitral valve interstitial cells in culture. (United States)

    Lester, W; Rosenthal, A; Granton, B; Gotlieb, A I


    There are connective tissue cells present within the interstitium of the heart valves. This study was designed to isolate and characterize mitral valve interstitial cells from the anterior leaflet of the mitral valve. Explants obtained from the distal part of the leaflet, having been scraped free of surface endocardial cells, were incubated in medium 199 supplemented with 10% fetal bovine serum. Cells grew out of the explant after 3 to 5 days and by 3 weeks these cells were harvested and passaged. Passages 1 to 22 were characterized in several explant sets. The cells showed a growth pattern reminiscent of fibroblasts. Growth was dependent on serum concentration. Cytoskeletal localization of actin and myosin showed prominent stress fibers. Ultrastructural studies showed many elongated cells with prominent stress fibers and some gap junctions and few adherens junctions. There were as well cells with fewer stress fibers containing prominent Golgi complex and dilated endoplasmic reticulum. In the multilayered superconfluent cultures, the former cells tended to be on the substratum of the dish or surface of the multilayered culture, whereas the latter was generally located within the layer of cells. Extracellular matrix was prominent in superconfluent cultures, often within the layers as well. Labeling of the cells with antibody HHF 35 (Tsukada T, Tippens D, Gordon D, Ross R, Gown AM: Am J Pathol 126:51, 1987), which recognizes smooth muscle cell actin, showed prominent staining of the elongated stress fiber-containing cells and much less in the secretory type cells. These studies show that interstitial mitral valve cells can be grown in culture and that either two different cell types or one cell type with two phenotypic expressions is present in culture.

  7. [Stem Cells in the Brain of Mammals and Human: Fundamental and Applied Aspects]. (United States)

    Aleksandrova, M A; Marey, M V


    Brain stem cells represent an extremely intriguing phenomenon. The aim of our review is to present an integrity vision of their role in the brain of mammals and humans, and their clinical perspectives. Over last two decades, investigations of biology of the neural stem cells produced significant changes in general knowledge about the processes of development and functioning of the brain. Researches on the cellular and molecular mechanisms of NSC differentiation and behavior led to new understanding of their involvement in learning and memory. In the regenerative medicine, original therapeutic approaches to neurodegenerative brain diseases have been elaborated due to fundamental achievements in this field. They are based on specific regenerative potential of neural stem cells and progenitor cells, which possess the ability to replace dead cells and express crucially significant biologically active factors that are missing in the pathological brain. For the needs of cell substitution therapy in the neural diseases, adequate methods of maintaining stem cells in culture and their differentiation into different types of neurons and glial cells, have been developed currently. The success of modern cellular technologies has significantly expanded the range of cells used for cell therapy. The near future may bring new perspective and distinct progress in brain cell therapy due to optimizing the cells types most promising for medical needs.

  8. Poststroke Cell Therapy of the Aged Brain

    Directory of Open Access Journals (Sweden)

    Aurel Popa-Wagner


    Full Text Available During aging, many neurodegenerative disorders are associated with reduced neurogenesis and a decline in the proliferation of stem/progenitor cells. The development of the stem cell (SC, the regenerative therapy field, gained tremendous expectations in the diseases that suffer from the lack of treatment options. Stem cell based therapy is a promising approach to promote neuroregeneration after brain injury and can be potentiated when combined with supportive pharmacological drug treatment, especially in the aged. However, the mechanism of action for a particular grafted cell type, the optimal delivery route, doses, or time window of administration after lesion is still under debate. Today, it is proved that these protections are most likely due to modulatory mechanisms rather than the expected cell replacement. Our group proved that important differences appear in the aged brain compared with young one, that is, the accelerated progression of ischemic area, or the delayed initiation of neurological recovery. In this light, these age-related aspects should be carefully evaluated in the clinical translation of neurorestorative therapies. This review is focused on the current perspectives and suitable sources of stem cells (SCs, mechanisms of action, and the most efficient delivery routes in neurorestoration therapies in the poststroke aged environment.

  9. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

    Directory of Open Access Journals (Sweden)

    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  10. Affinity (tropism) of caprine arthritis encephalitis virus for brain cells. (United States)

    Adebayo, I A; Olaleye, O D; Awoniyi, T A M


    One of the constraints in unraveling the mysteries blurring the advancement of research in the quest to totally put HIV problems under control is getting the appropriate animal model that would truly simulate human cases. This problem is more apparent in studies involving the central nervous system. Consequently, a viable animal model to generate information for the production of drugs and vaccines for the prevention and or control of lentiviral induced dementia in affected host animals is pertinent and vital. In this study, explant cultures prepared from the brain of new-born goat-kid were infected with CaprineArthritis Encephalitis (CAE) virus- a retrovirus affecting goats. The specific brain cell types infected by the (CAE) virus were determined using reverse-transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM techniques). TEM showed that in 85 - 90% cases, microglia were the cells specifically infected by the virus. Amplification of the genomic sequence of the envelope and the gag genes by RT-PCR confirmed the presence of CAEV proviral DNA in the brain cells of affected animals. No productive infection of the astrocytes was observed. The results of this study showed a lot of similarities in the tropism of CAE virus infection of goat brain cells to that of HIV infection in humans thus suggesting the potential usefulness of the caprine model for the study of HIV neuropathology. The goat model system as a non-primate model therefore could be more adaptable as a simple animal model than primate models with their complexity of anthropological, environmental and safety problems.

  11. Biological effect of velvet antler polypeptides on neural stem cells from embryonic rat brain

    Institute of Scientific and Technical Information of China (English)

    LU Lai-jin; CHEN Lei; MENG Xiao-ting; YANG Fan; ZHANG Zhi-xin; CHEN Dong


    Background Velvet antler polypeptides (VAPs), which are derived from the antler velvets, have been reported to maintain survival and promote growth and differentiation of neural cells and, especially the development of neural tissues. This study was designed to explore the influence of VAPs on neural stem cells in vitro derived from embryonic rat brain. Methods Neural stem cells derived from E12-14 rat brain were isolated, cultured, and expanded for 7 days until neural stem cell aggregations and neurospheres were generated. The neurospheres were cultured under the condition of different concentration of VAPs followed by immunocytochemistry to detect the differentiation of neural stem cells. Results VAPs could remarkablely promote differentiation of neural stem cells and most neural stem cells were induced to differentiate towards the direction of neurons under certain concentration of VAPs.Conclusion Neural stem cells can be successfully induced into neurons by VAPs in vitro, which could provide a basis for regeneration of the nervous system.

  12. Eye extract improves cell migration out of lymphoid organ explants of L. vannamei and viability of the primary cell cultures. (United States)

    Li, Wenfeng; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans


    Since no cell line from shrimp has been established up till now, an optimization of the primary cell culture protocol is necessary. In this context, the effect of extracts (supernatant of a 1:50 (w/v) suspension) from different shrimp organs (muscle, brain, ganglia, eyestalk, ovary, and eye) on the performance of primary lymphoid cell cultures was evaluated. Ten percent of eye extract and 3% of ovary extract enhanced maximally the migration and survival of cells of the lymphoid organ of Litopenaeus vannamei significantly at 48, 96, and 144 h post seeding. Extracts from the eyestalk (10%), muscle (10%), and brain (1%) significantly promoted the migration and survival of cells at 48 and 96 h post seeding but not anymore at 144 h post seeding. In conclusion, it may be advised to add 10% of eye extract or 3% of ovary extract to cells for the maximal health of primary cell cultures from the lymphoid organ of L. vannamei.

  13. Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro. (United States)

    Gianinazzi, C; Schild, M; Müller, N; Leib, S L; Simon, F; Nuñez, S; Joss, P; Gottstein, B


    The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

  14. Parvalbumin immunoreactivity is enhanced by brain-derived neurotrophic factor in organotypic cultures of rat retina. (United States)

    Rickman, D W


    The rodent retina undergoes considerable postnatal neurogenesis and phenotypic differentiation, and it is likely that diffusible neurotrophic factors contribute to this development and to the subsequent formation of functional retinal circuitry. Accordingly, perturbation of specific neurotrophin ligand-receptor interactions has provided valuable information as to the fundamental processes underlying this development. In the present studies we have built upon our previous observation that suppression of expression of trk(B), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), in the postnatal rat retina results in the alteration of a specific interneuron in the rod pathway-the parvalbumin (PV)-immunoreactive AII amacrine cell. Here, we isolated retinas from newborn rats and maintained them in organotypic culture for up to 14 days (approximating the time of eye opening, in vivo) in the presence of individual neurotrophins [BDNF or nerve growth factor (NGF)]. We then examined histological sections of cultures for PV immunoreactivity. In control cultures, only sparse PV-immunostained cells were observed. In cultures supplemented with NGF, numerous lightly immunostained somata were present in the inner nuclear layer (INL) at the border of the inner plexiform layer (IPL). Many of these cells had rudimentary dendritic arborizations in the IPL. Cultures supplemented with BDNF displayed numerous well-immunostained somata at the INL/IPL border that gave rise to elaborate dendritic arborizations that approximated the morphology of mature AII amacrine cells in vivo. These observations indicate that neurotrophins have specific effects upon the neurochemical and, perhaps, morphological differentiation of an important interneuron in a specific functional retinal circuit.

  15. Culture and transfection of axolotl cells. (United States)

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane


    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration.

  16. Wound Coverage by Cultured Skin Cells (United States)


    and spread. 6 We later coated collagen sponges with human or porcine plasma. Although this coating improved the plating of epidermal cells, it did not...healing by cultured epidermal grafts, we have found that: - We were able to grow epidermal cells on collapsed collagen sponges . As a result, we can create...plastic. Epidermal cells grown on collagen sponges formed four to five layers of nucleated cells, compared to only one layer on plastic surfaces. The use of

  17. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))


    Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

  18. Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro. (United States)

    Danielyan, K E; Abramyan, R A; Galoyan, A A; Kevorkian, G A


    We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

  19. Breast Cancer Cells May Change When They Spread to Brain (United States)

    ... page: Breast Cancer Cells May Change When They Spread to Brain: ... 2016 WEDNESDAY, Dec. 7, 2016 (HealthDay News) -- When breast cancer spreads to the brain, important molecular changes may ...

  20. Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes. (United States)

    Föllmann, W; Weber, S; Birkner, S


    Epithelial cells from bovine colon were isolated by mechanical preparation combined with an enzymatic digestion from colon specimens derived from freshly slaughtered animals. After digestion with collagenase I, the isolated tissue was centrifuged on a 2% D-sorbitol gradient to separate epithelial crypts which were seeded in collagen I-coated culture flasks. By using colon crypts and omitting the seeding of single cells a contamination by fibroblasts was prevented. The cells proliferated under the chosen culture conditions and formed monolayer cultures which were maintained for several weeks, including subcultivation steps. A population doubling time of about 21 hr was estimated in the log phase of the corresponding growth curve. During the culture period the cells were characterized morphologically and enzymatically. By using antibodies against cytokeratine 7 and 13 the isolated cells were identified as cells of epithelial origin. Antibodies against vimentin served as negative control. Morphological features such as microvilli, desmosomes and tight junctions, which demonstrated the ability of the cultured cells to restore an epithelial like monolayer, were shown by ultrastructural investigations. The preservation of the secretory function of the cultured cells was demonstrated by mucine cytochemistry with alcian blue staining. A stable expression of enzyme activities over a period of 6 days in culture occurred for gamma-glutamyltranspeptidase, acid phosphatase and NADH-dehydrogenase activity under the chosen culture conditions. Activity of alkaline phosphatase decreased to about 50% of basal value after 6 days in culture. Preliminary estimations of the metabolic competence of these cells revealed cytochrome P450 1A1-associated EROD activity in freshly isolated cells which was stable over 5 days in cultured cells. Then activity decreased completely. This culture system with primary epithelial cells from the colon will be used further as a model for the colon

  1. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.


    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  2. Human cell culture in a space bioreactor (United States)

    Morrison, Dennis R.


    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  3. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.


    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific productivit

  4. Pitfalls in cell culture work with xanthohumol. (United States)

    Motyl, M; Kraus, B; Heilmann, J


    Xanthohumol, the most abundant prenylated chalcone in hop (Humulus lupulus L.) cones, is well known to exert several promising pharmacological activities in vitro and in vivo. Among these, the chemopreventive, anti-inflammatory and anti-cancer effects are probably the most interesting. As xanthohumol is hardly soluble in water and able to undergo conversion to isoxanthohumol we determined several handling characteristics for cell culture work with this compound. Recovery experiments revealed that working with xanthohumol under cell culture conditions requires a minimal amount of 10% FCS to increase its solubility to reasonable concentrations (-50-75 micromol/l) for pharmacological in vitro tests. Additionally, more than 50% of xanthohumol can be absorbed to various plastic materials routinely used in the cell culture using FCS concentrations below 10%. In contrast, experiments using fluorescence microscopy in living cells revealed that detection of cellular intake of xanthohumol is hampered by concentrations above 1% FCS.

  5. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures (United States)

    Stampfer, Martha R; Garbe, James C


    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  6. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.


    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  7. Neural cell co-culture induced differentiation of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Nailong Yang; Lili Xu; Fen Yang


    BACKGROUND: It has been previously demonstrated that the neural cell microenvironment has the ability to induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into the neural cells.OBJECTIVE: To establish a co-culture system of human BMSCs and neural cells, and to observe effects of this co-culture system on differentiation of human BMSCs into neural cells.DESIGN, TIME AND SETTING: A comparative observation experiment, performed at the Center Laboratory of the Affiliated Hospital of Medical College Qingdao University from October 2006 to December 2007.MATERIALS: Neural cells were obtained from human fetal brain tissue. BMSCs were harvested from female patients that underwent autonomous stem cell transplantation.METHODS: BMSCs in the co-culture group consisted of BMSCs and third passage neural cells. BMSCs in the control group were solely cultured in vitro.MAIN OUTCOME MEASURES: Morphological changes of BMSCs were observed, and expression of the neuronal specific marker, neuron-specific enolase (NSE), was analyzed by immunofluorescence staining after4-5-day co-culture.RESULTS: The number of neural cells in the co-culture group increased and the cells spread on the culture bottle surface. Radial dendrite formed and connected with each other. NSE-immunoreactive cells were also detected. The positive ratio of NSE-positive cells reached (32.7±11.5)%, with morphological characteristics similar to neuronal cells. Human BMSCs did not express NSE in the control group.CONCLUSION: The microenvironment provided by neurons induced differentiation of BMSCs into neuronal-like cells.

  8. Balb/C鼠尾胶原与大鼠尾胶原对新生Balb/C鼠脑细胞培养的影响%Effects of Balb/C mouse tail collagen and rat tail collagen on newborn Balb/C mouse brain cells in culture

    Institute of Scientific and Technical Information of China (English)

    赵兰娟; 龚蕴贞; 林维芬; 胡涛; 张咏南


    目的确定鼠脑细胞体外培养的适宜生长基质。方法以Balb/C鼠尾胶原与大鼠尾胶原作为生长基质,培养新生Balb/C鼠脑细胞。结果 Balb/C鼠尾胶原有利于神经元的生存、生长与分化,而大鼠尾胶原则利于星形胶质细胞的生存、生长、分化与增殖。结论 2种鼠尾胶原均为培养鼠脑细胞适宜的生长基质,培养1个月左右宜用Balb/C鼠尾胶原而长期培养则宜选择大鼠尾胶原。%Objective To determine the suitable growth substrate for brain cells in culture. Methods Two kinds of collagen from Balb/C mouse tail and rat tail respectively as growth substrate was applied to culturing newborn Balb/C mouse brain cells. Results Balb/C mouse tail collagen was suitable to the survival, growth, differentiation of neurons, whereas rat tail collagen those of astrocytes. Conclusion Two kinds of collagen are suitable as growth substrate of neuron culture. Balb/C mouse tail collagen may be applied as growth substrate for short-term cultures, whereas rat tail collagen may be adopted for long-term cultures.

  9. A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays. (United States)

    Sandén, Emma; Eberstål, Sofia; Visse, Edward; Siesjö, Peter; Darabi, Anna


    In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.

  10. Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells. (United States)

    Nakagomi, Takayuki; Kubo, Shuji; Nakano-Doi, Akiko; Sakuma, Rika; Lu, Shan; Narita, Aya; Kawahara, Maiko; Taguchi, Akihiko; Matsuyama, Tomohiro


    Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

  11. 21 CFR 864.2280 - Cultured animal and human cells. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  12. Cell culture experiments planned for the space bioreactor (United States)

    Morrison, Dennis R.; Cross, John H.


    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  13. Pinoresinol from Ipomoea cairica cell cultures. (United States)

    Páska, Csilla; Innocenti, Gabbriella; Ferlin, Mariagrazia; Kunvári, Mónika; László, Miklós


    Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family. Pinoresinol was found to have antioxidant and Ca2+ antagonist properties. As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I. cairica cultures, as well as we continued investigations on lignans' response to optimization.

  14. Human brain endothelial cells endeavor to immunoregulate CD8 T cells via PD-1 ligand expression in multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Pittet Camille L


    Full Text Available Abstract Background Multiple sclerosis (MS, an inflammatory disease of the central nervous system (CNS, is characterized by blood-brain barrier (BBB disruption and massive infiltration of activated immune cells. Engagement of programmed cell death-1 (PD-1 expressed on activated T cells with its ligands (PD-L1 and PD-L2 suppresses T cell responses. We recently demonstrated in MS lesions elevated PD-L1 expression by glial cells and absence of PD-1 on many infiltrating CD8 T cells. We have now investigated whether human brain endothelial cells (HBECs, which maintain the BBB, can express PD-L1 or PD-L2 and thereby modulate T cells. Methods We used primary cultures of HBECs isolated from non-tumoral CNS tissue either under basal or inflamed conditions. We assessed the expression of PD-L1 and PD-L2 using qPCR and flow cytometry. Human CD8 T cells were isolated from peripheral blood of healthy donors and co-cultured with HBECs. Following co-culture with HBECs, proliferation and cytokine production by human CD8 T cells were measured by flow cytometry whereas transmigration was determined using a well established in vitro model of the BBB. The functional impact of PD-L1 and PD-L2 provided by HBECs was determined using blocking antibodies. We performed immunohistochemistry for the detection of PD-L1 or PD-L2 concurrently with caveolin-1 (a cell specific marker for endothelial cells on post-mortem human brain tissues obtained from MS patients and normal controls. Results Under basal culture conditions, PD-L2 is expressed on HBECs, whilst PD-L1 is not detected. Both ligands are up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 leads to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increases CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions does not express detectable PD-L1; in contrast, all blood vessels in normal

  15. Long term ex vivo culturing of Drosophila brain as a method to live image pupal brains: insights into the cellular mechanisms of neuronal remodeling. (United States)

    Rabinovich, Dana; Mayseless, Oded; Schuldiner, Oren


    Holometabolous insects, including Drosophila melanogaster, undergo complete metamorphosis that includes a pupal stage. During metamorphosis, the Drosophila nervous system undergoes massive remodeling and growth, that include cell death and large-scale axon and synapse elimination as well as neurogenesis, developmental axon regrowth, and formation of new connections. Neuronal remodeling is an essential step in the development of vertebrate and invertebrate nervous systems. Research on the stereotypic remodeling of Drosophila mushroom body (MB) γ neurons has contributed to our knowledge of the molecular mechanisms of remodeling but our knowledge of the cellular mechanisms remain poorly understood. A major hurdle in understanding various dynamic processes that occur during metamorphosis is the lack of time-lapse resolution. The pupal case and opaque fat bodies that enwrap the central nervous system (CNS) make live-imaging of the central brain in-vivo impossible. We have established an ex vivo long-term brain culture system that supports the development and neuronal remodeling of pupal brains. By optimizing culture conditions and dissection protocols, we have observed development in culture at kinetics similar to what occurs in vivo. Using this new method, we have obtained the first time-lapse sequence of MB γ neurons undergoing remodeling in up to a single cell resolution. We found that axon pruning is initiated by blebbing, followed by one-two nicks that seem to initiate a more widely spread axon fragmentation. As such, we have set up some of the tools and methodologies needed for further exploration of the cellular mechanisms of neuronal remodeling, not limited to the MB. The long-term ex vivo brain culture system that we report here could be used to study dynamic aspects of neurodevelopment of any Drosophila neuron.

  16. The effect of beta-turn structure on the permeation of peptides across monolayers of bovine brain microvessel endothelial cells

    DEFF Research Database (Denmark)

    Sorensen, M; Steenberg, B; Knipp, G T;


    PURPOSE: To investigate the effects of the beta-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). METHODS: The effective...

  17. General overview of neuronal cell culture. (United States)

    Gordon, Jennifer; Amini, Shohreh; White, Martyn K


    In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

  18. Wnt-Dependent Control of Cell Polarity in Cultured Cells. (United States)

    Runkle, Kristin B; Witze, Eric S


    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways.

  19. Bioactive form of resveratrol in glioblastoma cells and its safety for normal brain cells

    Directory of Open Access Journals (Sweden)

    Xiao-Hong Shu


    Full Text Available ABSTRACTBackground: Resveratrol, a plant polyphenol existing in grapes and many other natural foods, possesses a wide range of biological activities including cancer prevention. It has been recognized that resveratrol is intracellularly biotransformed to different metabolites, but no direct evidence has been available to ascertain its bioactive form because of the difficulty to maintain resveratrol unmetabolized in vivo or in vitro. It would be therefore worthwhile to elucidate the potential therapeutic implications of resveratrol metabolism using a reliable resveratrol-sensitive cancer cells.Objective: To identify the real biological form of trans-resveratrol and to evaluate the safety of the effective anticancer dose of resveratrol for the normal brain cells.Methods: The samples were prepared from the condition media and cell lysates of human glioblastoma U251 cells, and were purified by solid phase extraction (SPE. The samples were subjected to high performance liquid chromatography (HPLC and liquid chromatography/tandem mass spectrometry (LC/MS analysis. According to the metabolite(s, trans-resveratrol was biotransformed in vitro by the method described elsewhere, and the resulting solution was used to treat U251 cells. Meanwhile, the responses of U251 and primarily cultured rat normal brain cells (glial cells and neurons to 100μM trans-resveratrol were evaluated by multiple experimental methods.Results: The results revealed that resveratrol monosulfate was the major metabolite in U251 cells. About half fraction of resveratrol monosulfate was prepared in vitro and this trans-resveratrol and resveratrol monosulfate mixture showed little inhibitory effect on U251 cells. It is also found that rat primary brain cells (PBCs not only resist 100μM but also tolerate as high as 200μM resveratrol treatment.Conclusions: Our study thus demonstrated that trans-resveratrol was the bioactive form in glioblastoma cells and, therefore, the biotransforming

  20. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor (United States)

    Parks, Kelsey


    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  1. 3D culture for cardiac cells. (United States)

    Zuppinger, Christian


    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  2. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)


    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  3. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan


    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our focus...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....

  4. Nicotine-induced alterations in the expression of nicotinic receptors in primary cultures from human prenatal brain. (United States)

    Hellström-Lindahl, E; Seiger A; Kjaeldgaard, A; Nordberg, A


    The nicotinic receptor proteins and gene transcripts for the different nicotinic receptor subunits exist in human prenatal brain already at 4-5 weeks of gestation. The early presence of nicotinic receptors suggests an important role for these receptors in modulating dendritic outgrowth, establishment of neuronal connections and synaptogenesis during development. When measurements of nicotinic receptors using [(3)H]epibatidine (labelling both the alpha3 and alpha4 subtype) and [(3)H]cytisine (labelling the alpha4 subtype) were performed in intact cells from the cortex, subcortical forebrain and mesencephalon (7.5-11 weeks of gestation), the highest specific binding for both ligands was detected in cells from mesencephalon, followed by subcortical forebrain and cortex. The effects of nicotine exposure were studied in primary cultures of prenatal brain (7.5-11 weeks of gestation). Treatment with nicotine (1-100 microM) for 3 days significantly increased the specific binding of [(3)H]epibatidine and [(3)H]cytisine in cortical cells but not in cells from subcortical forebrain and mesencephalon brain regions, indicating region-specific differences in the sensitivity to nicotine exposure. Relative quantification of mRNA showed that the expression of the nicotinic receptor subunits alpha3 and alpha7, but not alpha4, was increased in cortical cells after nicotine treatment. These findings support the assumption of a potential risk of disturbance in the functional role of nicotinic receptors during brain development as a consequence of maternal smoking during pregnancy.

  5. Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells. (United States)

    Braun, Doreen; Kinne, Anita; Bräuer, Anja U; Sapin, Remy; Klein, Marc O; Köhrle, Josef; Wirth, Eva K; Schweizer, Ulrich


    Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan-Herndon-Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L-type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8-deficient patients. Functional T(3)- and T(4)-transport assays into primary astrocytes showed K(M) values of 4.2 and 3.7 μM for T(3) and T(4). Pharmacological inhibition of L-type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T(3) uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T(3) uptake further suggesting the cooperative activity of several T(3) transporters in astrocytes.

  6. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi


    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  7. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.


    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  8. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo

    Directory of Open Access Journals (Sweden)

    Zhiyuan Zou


    Full Text Available Cell penetrating peptides (CPPs are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26lacZ, we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  9. Optogenetic control of human neurons in organotypic brain cultures

    DEFF Research Database (Denmark)

    Andersson, My; Avaliani, Natalia; Svensson, Andreas


    Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof......-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies....

  10. Dynamic cell culture system (7-IML-1) (United States)

    Cogoli, Augusto


    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  11. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions. (United States)

    Stachniak, Tevye J E; Bourque, Charles W


    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  12. Correlation of cell apoptosis with brain edema and elevated intracranial pressure in traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    YANG Xiao-feng; LIU Wei-guo; SHEN Hong; GONG Jiang-biao; YU Jun; HU Wei-wei; L(U) Shi-ting; ZHENG Xiu-jue; FU Wei-ming


    Objective: To study the correlation between brain edema, elevated intracranial pressure (ICP) and cell apoptosis in traumatic brain injury (TBI). Methods: In this study, totally 42 rabbits in 7 groups were studied. Six of the animals were identified as a control group, and the remaining 36 animals were equally divided into 6 TBI groups. TBI models were produced by the modified method of Feeney. After the impact, ICP of each subject was recorded continuously by an ICP monitor until the animal was sacrificed at scheduled time. The apoptotic brain cells were detected by an terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Cerebral water content (CWC) was measured with a drying method and calculated according to the Elliott formula. Then, an analysis was conducted to determine the correlation between the count of apoptotic cells and the clinical pathological changes of the brain. Results: Apoptotic cell count began to increase 2 h after the impact, and reached its maximum about 3 days after the impact. The peak value of CWC and ICP appeared 1 day and 3 days after the impact, respectively. Apoptotic cell count had a positive correlation with CWC and ICP. Conclusions: In TBI, occurrence of brain edema and ICP increase might lead to apoptosis of brain cells. Any therapy which can relieve brain edema and/or decrease ICP would be able to reduce neuron apoptosis, thereby to attenuate the secondary brain damage.

  13. Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Erickson Michelle A


    Full Text Available Abstract Background Brain microvascular pericytes are important constituents of the neurovascular unit. These cells are physically the closest cells to the microvascular endothelial cells in brain capillaries. They significantly contribute to the induction and maintenance of the barrier functions of the blood-brain barrier. However, very little is known about their immune activities or their roles in neuroinflammation. Here, we focused on the immunological profile of brain pericytes in culture in the quiescent and immune-challenged state by studying their production of immune mediators such as nitric oxide (NO, cytokines, and chemokines. We also examined the effects of immune challenge on pericyte expression of low density lipoprotein receptor-related protein-1 (LRP-1, a protein involved in the processing of amyloid precursor protein and the brain-to-blood efflux of amyloid-β peptide. Methods Supernatants were collected from primary cultures of mouse brain pericytes. Release of nitric oxide (NO was measured by the Griess reaction and the level of S-nitrosylation of pericyte proteins measured with a modified "biotin-switch" method. Specific mitogen-activated protein kinase (MAPK pathway inhibitors were used to determine involvement of these pathways on NO production. Cytokines and chemokines were analyzed by multianalyte technology. The expression of both subunits of LRP-1 was analyzed by western blot. Results Lipopolysaccharide (LPS induced release of NO by pericytes in a dose-dependent manner that was mediated through MAPK pathways. Nitrative stress resulted in S-nitrosylation of cellular proteins. Eighteen of twenty-three cytokines measured were released constitutively by pericytes or with stimulation by LPS, including interleukin (IL-12, IL-13, IL-9, IL-10, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, eotaxin, chemokine (C-C motif ligand (CCL-3, and CCL-4. Pericyte expressions of both subunits of

  14. Brain Cancer Stem Cells: Current Status on Glioblastoma Multiforme



    Glioblastoma multiforme (GBM), an aggressive brain tumor of astrocytic/neural stem cell origin, represents one of the most incurable cancers. GBM tumors are highly heterogeneous. However, most tumors contain a subpopulation of cells that display neural stem cell characteristics in vitro and that can generate a new brain tumor upon transplantation in mice. Hence, previously identified molecular pathways regulating neural stem cell biology were found to represent the cornerstone of GBM stem cel...

  15. Gadolinium uptake by brain cancer cells: Quantitative analysis with X-PEEM spectromicroscopy for cancer therapy (United States)

    De Stasio, Gelsomina; Gilbert, B.; Perfetti, P.; Margaritondo, G.; Mercanti, D.; Ciotti, M. T.; Casalbore, P.; Larocca, L. M.; Rinelli, A.; Pallini, R.


    We present the first X-PEEM spectromicroscopy semi-quantitative data, acquired on Gd in glioblastoma cell cultures from human brain cancer. The cells were treated with a Gd compound for the optimization of GdNCT (Gadolinium Neutron Capture Therapy). We analyzed the kinetics of Gd uptake as a function of exposure time, and verified that a quantitative analytical technique gives the same results as our MEPHISTO X-PEEM, demonstrating the feasibility of semi-quantitative spectromicroscopy.

  16. Ammonium accumulation and cell death in a rat 3D brain cell model of glutaric aciduria type I.

    Directory of Open Access Journals (Sweden)

    Paris Jafari

    Full Text Available Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i 3-OHGA causes the death of astrocytes, (ii deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.

  17. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    Directory of Open Access Journals (Sweden)

    Parvaneh Farzaneh


    Full Text Available One of the main problems in cell culture is mycoplasma infection. It can extensively affectcell physiology and metabolism. As the applications of cell culture increase in research,industrial production and cell therapy, more concerns about mycoplasma contaminationand detection will arise. This review will provide valuable information about: 1. the waysin which cells are contaminated and the frequency and source of mycoplasma species incell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importanceof mycoplasma tests in cell culture; 4. different methods to identify mycoplasmacontamination; 5. the consequences of mycoplasma contamination in cell culture and 6.available methods to eliminate mycoplasma contamination. Awareness about the sourcesof mycoplasma and pursuing aseptic techniques in cell culture along with reliable detectionmethods of mycoplasma contamination can provide an appropriate situation to preventmycoplasma contamination in cell culture.

  18. Are there fetal stem cells in the maternal brain?

    Institute of Scientific and Technical Information of China (English)

    Osman Demirhan; Necmi (C)ekin; Deniz Ta(s)temir; Erdal Tun(c); Ali irfan Güzel; Demet Meral; Bülent Demirbek


    Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother's brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby's stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

  19. The effect of apolipoprotein E4 on synchronous neural interactions in brain cultures. (United States)

    Christopoulos, Vassilios; Georgopoulos, Angeliki; Georgopoulos, Apostolos P


    In a previous study, we assessed the synchronous neural interactions (SNI) in a developing neural network in brain cultures on multielectrode arrays (Christopoulos et al. in J Neural Eng 9:046008, 2012). Here, we report on the effects of apolipoprotein E4 (apoE4) on these neural interactions. We carried out six experiments (five using rodent brain cultures and one using neuroblastoma cultures) in which we recorded local field potentials (LFP) from 59 sites for several days in vitro under the following conditions. In one experiment, we added to the culture media triglyceride (TG)-rich lipoproteins from a human subject with the apoE4/4 genotype, whereas in the other experiments, we added recombinant human apoE4. We found that SNI in the apoE4-treated cultures had higher coefficient of SNI variation, as compared to control cultures. These findings further document the role of SNI as a fundamental aspect of the dynamic organization of neural networks (Langheim et al. in Proc Natl Acad Sci USA 103:455-459, 2006. doi: 10.1073/pnas.0509623102 ; Georgopoulos et al. in J Neural Eng 4:349-355, 2007) and extend the effect of apoE4 on SNI (Leuthold et al. in Exp Brain Res 226:525-536, 2013) across different brain species (human, rodents), apoE source (TG-rich lipoproteins, recombinant), neural signals (MEG, LFP), and brain network (intact brain, developing brain in vitro). To our knowledge, this is the first study of the effects of apoE4 on neural network function in vitro.

  20. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi


    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  1. Bioengineered 3D Glial Cell Culture Systems and Applications for Neurodegeneration and Neuroinflammation. (United States)

    Watson, P Marc D; Kavanagh, Edel; Allenby, Gary; Vassey, Matthew


    Neurodegeneration and neuroinflammation are key features in a range of chronic central nervous system (CNS) diseases such as Alzheimer's and Parkinson's disease, as well as acute conditions like stroke and traumatic brain injury, for which there remains significant unmet clinical need. It is now well recognized that current cell culture methodologies are limited in their ability to recapitulate the cellular environment that is present in vivo, and there is a growing body of evidence to show that three-dimensional (3D) culture systems represent a more physiologically accurate model than traditional two-dimensional (2D) cultures. Given the complexity of the environment from which cells originate, and their various cell-cell and cell-matrix interactions, it is important to develop models that can be controlled and reproducible for drug discovery. 3D cell models have now been developed for almost all CNS cell types, including neurons, astrocytes, microglia, and oligodendrocyte cells. This review will highlight a number of current and emerging techniques for the culture of astrocytes and microglia, glial cell types with a critical role in neurodegenerative and neuroinflammatory conditions. We describe recent advances in glial cell culture using electrospun polymers and hydrogel macromolecules, and highlight how these novel culture environments influence astrocyte and microglial phenotypes in vitro, as compared to traditional 2D systems. These models will be explored to illuminate current trends in the techniques used to create 3D environments for application in research and drug discovery focused on astrocytes and microglial cells.

  2. Uptake and metabolism of iron oxide nanoparticles in brain cells. (United States)

    Petters, Charlotte; Irrsack, Ellen; Koch, Michael; Dringen, Ralf


    Magnetic iron oxide nanoparticles (IONPs) are used for various applications in biomedicine, for example as contrast agents in magnetic resonance imaging, for cell tracking and for anti-tumor treatment. However, IONPs are also known for their toxic effects on cells and tissues which are at least in part caused by iron-mediated radical formation and oxidative stress. The potential toxicity of IONPs is especially important concerning the use of IONPs for neurobiological applications as alterations in brain iron homeostasis are strongly connected with human neurodegenerative diseases. Since IONPs are able to enter the brain, potential adverse consequences of an exposure of brain cells to IONPs have to be considered. This article describes the pathways that allow IONPs to enter the brain and summarizes the current knowledge on the uptake, the metabolism and the toxicity of IONPs for the different types of brain cells in vitro and in vivo.





    Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesi...

  4. Embryo forming cells in carrot suspension cultures.


    Toonen, M.A.J.


    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  5. Mouse cell culture: methods and protocols


    Elvira M. Guerra Shinohara


    The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases), starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward ...

  6. Preliminary Study of Realistic Blast Impact on Cultured Brain Slices (United States)


    integral membrane protein belonging to the glutamate-gated ion channel family, whereas a triple blast insult caused a significant reduction in the...24 I.U. per mg), 5-units/ml penicillin , and 5-mg/l streptomycin. The prepared slice cultures were supplied with fresh HSM 24 h later (culture day 1... penicillin , and 5-mg/L streptomycin. Just prior to submerging the blast plate, a control plate of slices was flooded with warmed SFM to provide tissue

  7. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages. (United States)

    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A


    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  8. Adult human brain neural progenitor cells (NPCs and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

    Directory of Open Access Journals (Sweden)

    Thomas In-Hyeup Park

    Full Text Available The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia, and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs. These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting Ah

  9. Staphylococcal α-hemolysin is neurotoxic and causes lysis of brain cells in vivo and in vitro. (United States)

    Dahlberg, Daniel; Mariussen, Espen; Goverud, Ingeborg Løstegaard; Tønjum, Tone; Mæhlen, Jan; Antal, Ellen-Ann; Hassel, Bjørnar


    Formation of a bacterial brain abscess entails loss of brain cells and formation of pus. The mechanisms behind the cell loss are not fully understood. Staphylococcus aureus, a common cause of brain abscesses, produces various exotoxins, including α-hemolysin, which is an important factor in brain abscess formation. α-Hemolysin may cause cytolysis by forming pores in the plasma membrane of various eukaryotic cells. However, whether α-hemolysin causes lysis of brain cells is not known. Nor is it known whether α-hemolysin in the brain causes cell death through pore formation or by acting as a chemoattractant, recruiting leukocytes and causing inflammation. Here we show that α-hemolysin injected into rat brain causes cell damage and edema formation within 30 min. Cell damage was accompanied by an increase in extracellular concentrations of zinc, GABA, glutamate, and other amino acids, indicating plasma membrane damage, but leukocytic infiltration was not seen 0.5-12h after α-hemolysin injection. This was in contrast to injection of S. aureus, which triggered extensive infiltration with neutrophils within 8h. In vitro, α-hemolysin caused concentration-dependent lysis of isolated nerve endings and cultured astrocytes. We conclude that α-hemolysin contributes to the cell death inherent in staphylococcal brain abscess formation as a pore-forming neurotoxin.

  10. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.


    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  11. NMR imaging of cell phone radiation absorption in brain tissue


    Gultekin, David H.; Moeller, Lothar


    A method is described for measuring absorbed electromagnetic energy radiated from cell phone antennae into ex vivo brain tissue. NMR images the 3D thermal dynamics inside ex vivo bovine brain tissue and equivalent gel under exposure to power and irradiation time-varying radio frequency (RF) fields. The absorbed RF energy in brain tissue converts into Joule heat and affects the nuclear magnetic shielding and the Larmor precession. The resultant temperature increase is measured by the resonance...

  12. Cultured cells of the nervous system, including human neurones, in the study of the neuro-degenerative disorder, Alzheimer's disease: an overview. (United States)

    De Boni, U


    Human nervous-system cells in culture are a suitable model for the study of the degenerative changes associated with Alzheimer's disease. Alzheimer-diseased brain contains a factor which induces the formation of paired helical filaments (PHF) in cultured cells, similar to that seen in Alzheimer's disease. The excitotoxic amino acids, glutamate and aspartate, induce similar PHE formation in cultured cells. The neurotoxic element aluminium is present in high concentrations in the brain in several human neurological disorders, including Alzheimer's disease. In cultured-cell systems, aluminium interacts with acidic nuclear proteins, decreases steroid binding, produces a form of neurofibrillary degeneration and alters nucleoside metabolism.

  13. Insect cell culture in research: Indian scenario. (United States)

    Sudeep, A B; Mourya, D T; Mishra, A C


    Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies.

  14. Analysis of Neural Stem Cells from Human Cortical Brain Structures In Vitro. (United States)

    Aleksandrova, M A; Poltavtseva, R A; Marei, M V; Sukhikh, G T


    Comparative immunohistochemical analysis of the neocortex from human fetuses showed that neural stem and progenitor cells are present in the brain throughout the gestation period, at least from week 8 through 26. At the same time, neural stem cells from the first and second trimester fetuses differed by the distribution, morphology, growth, and quantity. Immunocytochemical analysis of neural stem cells derived from fetuses at different gestation terms and cultured under different conditions showed their differentiation capacity. Detailed analysis of neural stem cell populations derived from fetuses on gestation weeks 8-9, 18-20, and 26 expressing Lex/SSEA1 was performed.

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

    DEFF Research Database (Denmark)

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


    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...... for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added...... to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...

  16. Mesenchymal Stem Cell Transplantation Attenuates Brain Injury After Neonatal Stroke

    NARCIS (Netherlands)

    van Velthoven, Cindy T. J.; Sheldon, R. Ann; Kavelaars, Annemieke; Derugin, Nikita; Vexler, Zinaida S.; Willemen, Hanneke L. D. M.; Maas, Mirjam; Heijnen, Cobi J.; Ferriero, Donna M.


    Background and Purpose-Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulati

  17. A high-throughput cell-based method to predict the unbound drug fraction in the brain. (United States)

    Mateus, André; Matsson, Pär; Artursson, Per


    Optimization of drug efficacy in the brain requires understanding of the local exposure to unbound drug at the site of action. This relies on measurements of the unbound drug fraction (fu,brain), which currently requires access to brain tissue. Here, we present a novel methodology using homogenates of cultured cells for rapid estimation of fu,brain. In our setup, drug binding to human embryonic kidney cell (HEK293) homogenate was measured in a small-scale dialysis apparatus. To increase throughput, we combined drugs into cassettes for simultaneous measurement of multiple compounds. Our method estimated fu,brain with an average error of 1.9-fold. We propose that our simple method can be used as an inexpensive, easily available and high-throughput alternative to brain tissues excised from laboratory animals. Thereby, estimates of unbound drug exposure can now be implemented at a much earlier stage of the drug discovery process, when molecular property changes are easier to make.


    Directory of Open Access Journals (Sweden)



    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  19. How do culture media influence in vitro perivascular cell behavior? (United States)

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane


    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

  20. Mouse neural stem cells cultured in vitro and expressing an exogenous gene

    Institute of Scientific and Technical Information of China (English)


    Neural stem cells are the multipotential, self-re- newing cells in central nerve system, and play an essential role in the development and differentiation of nerve system. Neural stem cells can be used to treat the nerve system diseases, especially, the transplantation of neural stem cells to rescue the degenerated neural cells has become a very promising therapeutic way. We successfully cultured neural stem cells isolated from the brains of embryonic mice in vitro and determined their distribution in the E17 mice brains. The neural stem cells were transfected with adenoviral vector carrying GFP (green fluorescence protein) gene and then highly expressed the exogenous gene. It paves the way for gene therapy of degenerative nerve system diseases.

  1. Green tea may protect brain cells against Parkinson's

    Institute of Scientific and Technical Information of China (English)


    @@ Does the consumption of green tea, widely touted to have beneficial effects on health, also protect brain cells? As reported in the 15 December, 2007 issue of Biological Psychiatry, studies by CAS researchers indicate this may be the case.

  2. Sarcoma derived from cultured mesenchymal stem cells. (United States)

    Tolar, Jakub; Nauta, Alma J; Osborn, Mark J; Panoskaltsis Mortari, Angela; McElmurry, Ron T; Bell, Scott; Xia, Lily; Zhou, Ning; Riddle, Megan; Schroeder, Tania M; Westendorf, Jennifer J; McIvor, R Scott; Hogendoorn, Pancras C W; Szuhai, Karoly; Oseth, Leann; Hirsch, Betsy; Yant, Stephen R; Kay, Mark A; Peister, Alexandra; Prockop, Darwin J; Fibbe, Willem E; Blazar, Bruce R


    To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.


    Directory of Open Access Journals (Sweden)

    Kiselev K.V.


    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  4. Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models. (United States)

    Choi, Yoon Pyo; Lee, Joo Hyun; Gao, Ming-Qing; Kim, Baek Gil; Kang, Suki; Kim, Se Hoon; Cho, Nam Hoon


    Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6-siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.

  5. NMR imaging of cell phone radiation absorption in brain tissue. (United States)

    Gultekin, David H; Moeller, Lothar


    A method is described for measuring absorbed electromagnetic energy radiated from cell phone antennae into ex vivo brain tissue. NMR images the 3D thermal dynamics inside ex vivo bovine brain tissue and equivalent gel under exposure to power and irradiation time-varying radio frequency (RF) fields. The absorbed RF energy in brain tissue converts into Joule heat and affects the nuclear magnetic shielding and the Larmor precession. The resultant temperature increase is measured by the resonance frequency shift of hydrogen protons in brain tissue. This proposed application of NMR thermometry offers sufficient spatial and temporal resolution to characterize the hot spots from absorbed cell phone radiation in aqueous media and biological tissues. Specific absorption rate measurements averaged over 1 mg and 10 s in the brain tissue cover the total absorption volume. Reference measurements with fiber optic temperature sensors confirm the accuracy of the NMR thermometry.

  6. Cell Mediated Photothermal Therapy of Brain Tumors. (United States)

    Hirschberg, Henry; Madsen, Steen J


    Gold based nanoparticles with strong near infra-red (NIR) absorption are ideally suited for photothermal therapy (PTT) of brain tumors. The goal of PTT is to induce rapid heating in tumor tissues while minimizing thermal diffusion to normal brain. PTT efficacy is sensitively dependent on both nanoparticle concentration and distribution in tumor tissues. Nanoparticle delivery via passive approaches such as the enhanced permeability and retention (EPR) effect is unlikely to achieve sufficient nanoparticle concentrations throughout tumor volumes required for effective PTT. A simple approach for improving tumor biodsitribution of nanoparticles is the use of cellular delivery vehicles. Specifically, this review focuses on the use of monocytes/macrophages (Mo/Ma) as gold nanoparticle delivery vectors for PTT of brain tumors. Although the efficacy of this delivery approach has been demonstrated in both in vitro and animal PTT studies, its clinical potential for the treatment of brain tumors remains uncertain.

  7. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;


    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  8. Propagation of human germ stem cells in long-term culture

    Directory of Open Access Journals (Sweden)

    Abbas Khodadadi


    Full Text Available Background: Spermatogonial stem cells (SSCs, a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. Objective: The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs and improvement of presence of human Germ Stem Cells (hGSCs were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1, also known as Octamer-binding transcription factor 4 (Oct-4 and PLZF (Promyelocytic leukaemia zinc finger protein. Materials and Methods: Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin. Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. Results: hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. Conclusion: hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions.

  9. In vitro differentiation of cultured human CD34+ cells into astrocytes

    Directory of Open Access Journals (Sweden)

    Katari Venkatesh


    Full Text Available Background: Astrocytes are abundantly present as glial cells in the brain and play an important role in the regenerative processes. The possible role of stem cell derived astrocytes in the spinal cord injuries is possible related to their influence at the synaptic junctions. Aim: The present study is focused on in vitro differentiation of cultured human CD34+ cells into astrocytes. Materials and Methods: Granulocyte-colony stimulating factor mobilized human CD34+ cells were isolated from peripheral blood using apheresis method from a donor. These cells were further purified by fluorescence-activated cell sorting and cultured in Dulbecco′s modified eagle′s medium. Thus, cultured cells were induced with astrocyte defined medium (ADM and in the differentiated astrocytes serine/threonine protein kinases (STPK and glutamine synthetase (GLUL activities were estimated. The expression of glial fibrillary acidic protein (GFAP and GLUL were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR. Results: The cultured human CD34+ cells differentiated into astrocytes after 11 h of incubation in ADM. The RT-PCR experiment showed the expression of GLUL (1.5 kb and GFAP (2.9 kb in differentiated astrocytes. The high enzyme activities of GLUL and STPK in differentiated astrocytes compared with cultured human CD34+ cells confirmed astrocyte formation. Conclusion: In the present study, in vitro differentiation of stem cells with retinoic acid induction may result in the formation of astrocytes.

  10. Equipment for large-scale mammalian cell culture. (United States)

    Ozturk, Sadettin S


    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed.

  11. Programmed Necrosis: A Prominent Mechanism of Cell Death following Neonatal Brain Injury

    Directory of Open Access Journals (Sweden)

    Raul Chavez-Valdez


    Full Text Available Despite the introduction of therapeutic hypothermia, neonatal hypoxic ischemic (HI brain injury remains a common cause of developmental disability. Development of rational adjuvant therapies to hypothermia requires understanding of the pathways of cell death and survival modulated by HI. The conceptualization of the apoptosis-necrosis “continuum” in neonatal brain injury predicts mechanistic interactions between cell death and hydrid forms of cell death such as programmed or regulated necrosis. Many of the components of the signaling pathway regulating programmed necrosis have been studied previously in models of neonatal HI. In some of these investigations, they participate as part of the apoptotic pathways demonstrating clear overlap of programmed death pathways. Receptor interacting protein (RIP-1 is at the crossroads between types of cellular death and survival and RIP-1 kinase activity triggers formation of the necrosome (in complex with RIP-3 leading to programmed necrosis. Neuroprotection afforded by the blockade of RIP-1 kinase following neonatal HI suggests a role for programmed necrosis in the HI injury to the developing brain. Here, we briefly review the state of the knowledge about the mechanisms behind programmed necrosis in neonatal brain injury recognizing that a significant proportion of these data derive from experiments in cultured cell and some from in vivo adult animal models. There are still more questions than answers, yet the fascinating new perspectives provided by the understanding of programmed necrosis in the developing brain may lay the foundation for new therapies for neonatal HI.

  12. Growth hormone responsive neural precursor cells reside within the adult mammalian brain


    Blackmore, Daniel G.; Brent A. Reynolds; Golmohammadi, Mohammad G.; Large, Beatrice; Aguilar, Roberto M.; Haro, Luis; Waters, Michael J.; Rietze, Rodney L.


    The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice, while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large ...

  13. Anatomy, technology, art, and culture: toward a realistic perspective of the brain. (United States)

    Cavalcanti, Daniel D; Feindel, William; Goodrich, James T; Dagi, T Forcht; Prestigiacomo, Charles J; Preul, Mark C


    In the 15th century, brain illustration began to change from a schematic system that involved scant objective rendering of the brain, to accurate depictions based on anatomical dissections that demanded significant artistic talent. Notable examples of this innovation are the drawings of Leonardo da Vinci (1498-1504), Andreas Vesalius' association with the bottega of Titian to produce the drawings of Vesalius' De humani corporis fabrica (1543), and Christopher Wren's illustrations for Thomas Willis' Cerebri Anatome (1664). These works appeared during the Renaissance and Age of Enlightenment, when advances in brain imaging, or really brain rendering, reflected not only the abilities and dedications of the artists, but also the influences of important cultural and scientific factors. Anatomy and human dissection became popular social phenomena as well as scholarly pursuits, linked with the world of the fine arts. The working philosophy of these artists involved active participation in both anatomical study and illustration, and the belief that their discoveries of the natural world could best be communicated by rendering them in objective form (that is, with realistic perspective). From their studies emerged the beginning of contemporary brain imaging. In this article, the authors examine how the brain began to be imaged in realism within a cultural and scientific milieu that witnessed the emergence of anatomical dissection, the geometry of linear perspective, and the closer confluence of art and science.

  14. Recombinant Protein Production and Insect Cell Culture and Process (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)


    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  15. Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue. (United States)

    Wong, Fong Kuan; Haffner, Christiane; Huttner, Wieland B; Taverna, Elena


    This microinjection protocol allows the manipulation and tracking of neural stem and progenitor cells in tissue at single-cell resolution. We demonstrate how to apply microinjection to organotypic brain slices obtained from mice and ferrets; however, our technique is not limited to mouse and ferret embryos, but provides a means of introducing a wide variety of membrane-impermeable molecules (e.g., nucleic acids, proteins, hydrophilic compounds) into neural stem and progenitor cells of any developing mammalian brain. Microinjection experiments are conducted by using a phase-contrast microscope equipped with epifluorescence, a transjector and a micromanipulator. The procedure normally takes ∼2 h for an experienced researcher, and the entire protocol, including tissue processing, can be performed within 1 week. Thus, microinjection is a unique and versatile method for changing and tracking the fate of a cell in organotypic slice culture.

  16. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi


    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  17. Microfluidics and cancer analysis: cell separation, cell/tissue culture, cell mechanics, and integrated analysis systems. (United States)

    Pappas, Dimitri


    Among the growing number of tools available for cancer studies, microfluidic systems have emerged as a promising analytical tool to elucidate cancer cell and tumor function. Microfluidic methods to culture cells have created approaches to provide a range of environments from single-cell analysis to complex three-dimensional devices. In this review we discuss recent advances in tumor cell culture, cancer cell analysis, and advanced studies enabled by microfluidic systems.

  18. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan


    -defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

  19. Stem Cells and the Origin and Propagation of Brain Tumors



    In recent years there has been a flood of interest in the relationship between brain tumors and stem cells. Some investigators have focused on the sensitivity of normal stem cells to transformation, others have described phenotypic or functional similarities between tumor cells and stem cells, and still others have suggested that tumors contain a subpopulation of “cancer stem cells” that is crucial for tumor maintenance or propagation. While all these concepts are interesting and provide insi...

  20. A stable and reproducible human blood-brain barrier model derived from hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Romeo Cecchelli

    Full Text Available The human blood brain barrier (BBB is a selective barrier formed by human brain endothelial cells (hBECs, which is important to ensure adequate neuronal function and protect the central nervous system (CNS from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

  1. Cell proliferation and neurogenesis in adult mouse brain.

    Directory of Open Access Journals (Sweden)

    Olivia L Bordiuk

    Full Text Available Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2'-deoxyuridine (EdU to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ, and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain.

  2. Live-cell imaging to detect phosphatidylserine externalization in brain endothelial cells exposed to ionizing radiation: implications for the treatment of brain arteriovenous malformations. (United States)

    Zhao, Zhenjun; Johnson, Michael S; Chen, Biyi; Grace, Michael; Ukath, Jaysree; Lee, Vivienne S; McRobb, Lucinda S; Sedger, Lisa M; Stoodley, Marcus A


    OBJECT Stereotactic radiosurgery (SRS) is an established intervention for brain arteriovenous malformations (AVMs). The processes of AVM vessel occlusion after SRS are poorly understood. To improve SRS efficacy, it is important to understand the cellular response of blood vessels to radiation. The molecular changes on the surface of AVM endothelial cells after irradiation may also be used for vascular targeting. This study investigates radiation-induced externalization of phosphatidylserine (PS) on endothelial cells using live-cell imaging. METHODS An immortalized cell line generated from mouse brain endothelium, bEnd.3 cells, was cultured and irradiated at different radiation doses using a linear accelerator. PS externalization in the cells was subsequently visualized using polarity-sensitive indicator of viability and apoptosis (pSIVA)-IANBD, a polarity-sensitive probe. Live-cell imaging was used to monitor PS externalization in real time. The effects of radiation on the cell cycle of bEnd.3 cells were also examined by flow cytometry. RESULTS Ionizing radiation effects are dose dependent. Reduction in the cell proliferation rate was observed after exposure to 5 Gy radiation, whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with cells treated with sham radiation, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA-positive cells were significantly higher (p = 0.04) 24 hours after treatment in the cultures that received 25- and 15-Gy doses of radiation. This effect was sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation controls at any time points (p > 0.15). Flow cytometric analysis data indicate that irradiation induced growth arrest of bEnd.3 cells, with cells accumulating in the G2 phase of the cell cycle. CONCLUSIONS Ionizing radiation

  3. Sex steroids and their receptors: molecular actions on brain cells. (United States)

    Mannella, Paolo; Simoncini, Tommaso


    Sex steroids exert actions of paramount importance on brain cells. They contribute to shape the central nervous system during embryo development. They modulate the formation and the turnover of the interconnections between neurons. They control the function of glial cells. And they do it through a signaling machinery that is apparently simple, but that hides a level of complexity that has been unveiled only in part. Different receptor isoforms, different interactions between receptors and co-regulators, chains of events originating at the cell membrane and leading to effects in the nucleus (or the other way around) all interact to determine selective modulations of brain cells. All these actions end up in phenomenal effects on brain function that change through adolescence, pregnancy, adulthood, up to menopause and ageing. Many of these actions are relevant for degenerative processes and research may offer soon new strategies to counteract these diseases.

  4. Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development

    Directory of Open Access Journals (Sweden)

    Bello Bruno C


    Full Text Available Abstract Background In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors. Results Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells. Conclusion Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.

  5. Three-dimensional hydrogel cell culture systems for modeling neural tissue (United States)

    Frampton, John

    Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was

  6. Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection


    Di Giovanni, George D.; LeChevallier, Mark W.


    A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-Q...

  7. Mesenchymal Stem Cells in the Treatment of Traumatic Brain Injury (United States)

    Hasan, Anwarul; Deeb, George; Rahal, Rahaf; Atwi, Khairallah; Mondello, Stefania; Marei, Hany Elsayed; Gali, Amr; Sleiman, Eliana


    Traumatic brain injury (TBI) is characterized by a disruption in the normal function of the brain due to an injury following a trauma, which can potentially cause severe physical, cognitive, and emotional impairment. The primary insult to the brain initiates secondary injury cascades consisting of multiple complex biochemical responses of the brain that significantly influence the overall severity of the brain damage and clinical sequelae. The use of mesenchymal stem cells (MSCs) offers huge potential for application in the treatment of TBI. MSCs have immunosuppressive properties that reduce inflammation in injured tissue. As such, they could be used to modulate the secondary mechanisms of injury and halt the progression of the secondary insult in the brain after injury. Particularly, MSCs are capable of secreting growth factors that facilitate the regrowth of neurons in the brain. The relative abundance of harvest sources of MSCs also makes them particularly appealing. Recently, numerous studies have investigated the effects of infusion of MSCs into animal models of TBI. The results have shown significant improvement in the motor function of the damaged brain tissues. In this review, we summarize the recent advances in the application of MSCs in the treatment of TBI. The review starts with a brief introduction of the pathophysiology of TBI, followed by the biology of MSCs, and the application of MSCs in TBI treatment. The challenges associated with the application of MSCs in the treatment of TBI and strategies to address those challenges in the future have also been discussed.

  8. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man


    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  9. Growth of the Pittsburgh Pneumonia Agent in Animal Cell Cultures


    Rinaldo, Charles R.; Pasculle, A. William; Myerowitz, Richard L.; Gress, Francis M.; Dowling, John N.


    Pittsburgh pneumonia agent (Legionella micdadei) grew in monkey, chicken, and human cell cultures. Pittsburgh pneumonia agent grew predominantly in the cytoplasm, resulting in a nonfocal, mild cytopathic effect.

  10. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model. (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami


    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.


    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia


    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

  12. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs......, these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...

  13. Statistical study of biomechanics of living brain cells during growth and maturation on artificial substrates. (United States)

    Chen, La; Li, Wenfang; Maybeck, Vanessa; Offenhäusser, Andreas; Krause, Hans-Joachim


    There is increasing evidence that mechanical issues play a vital role in neuron growth and brain development. The importance of this grows as novel devices, whose material properties differ from cells, are increasingly implanted in the body. In this work, we studied the mechanical properties of rat brain cells over time and on different materials by using a high throughput magnetic tweezers system. It was found that the elastic moduli of both neurite and soma in networked neurons increased with growth. However, neurites at DIV4 exhibited a relatively high stiffness, which could be ascribed to the high outgrowth tension. The power-law exponents (viscoelasticity) of both neurites and somas of neurons decreased with culture time. On the other hand, the stiffness of glial cells also increased with maturity. Furthermore, both neurites and glia become softer when cultured on compliant substrates. Especially, the glial cells cultured on a soft substrate obviously showed a less dense and more porous actin and GFAP mesh. In addition, the viscoelasticity of both neurites and glia did not show a significant dependence on the substrates' stiffness.


    Ryzhak, A P; Chalisova, N I; Lin'kova, N S; Khalimov, R I; Ryzhak, G A; Zhekalov, A N


    A comparative study of polypeptides extracted from the tissues of calves: Cortexin (from brain cortex), Epinorm (from pineal gland), Ventvil (from liver), Prostatilen (from prostate), Thymalin (from thymus), Chelohart (from heart), Chondrolux (from cartilage) on the relevant organotypic tissue cultures of young and old rats, in concentration 0,01-100 ng/ml was performed. Polypeptides specifically stimulated "young" and "old" cell cultures growth in concentration 20-50 ng/ml. This effect correlates with increasing of PCNA and decreasing of p53 expression in brain cortex, pineal gland, liver, prostate, heart, cartilage. Moreover, Thymalin activated CD5, CD20 expression--markers of B-cells differentiation. These data show that polypeptides isolated from different tissues have selective molecular activity on the regeneration of suitable tissues in aging.


    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.


    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  16. An Optically Controlled 3D Cell Culturing System

    Directory of Open Access Journals (Sweden)

    Kelly S. Ishii


    Full Text Available A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability.

  17. Aeroponics for the culture of organisms, tissues and cells. (United States)

    Weathers, P J; Zobel, R W


    Characteristics of aeroponics are discussed. Contrast is made, where appropriate, with hydroponics and aero-hydroponics as applies to research and commercial applications of nutrient mist technology. Topics include whole plants, plant tissue cultures, cell and microbial cultures, and animal tissue cultures with regard to operational considerations (moisture, temperature, minerals, gaseous atmosphere) and design of apparati.

  18. Challenges in providing culturally-competent care to patients with metastatic brain tumours and their families. (United States)

    Longo, Lianne; Slater, Serena


    Being diagnosed with a metastatic brain tumour can be devastating as it is characterized by very low cure rates, as well as significant morbidity and mortality. Given the poor life expectancy and progressive disability that ensues, patients and family members experience much turmoil, which includes losses that bring about changes to family roles, routines and relationships. Crisis and conflict are common during such major disruptions to a family system, as individual members attempt to make sense of the illness experience based on cultural and spiritual beliefs, past experiences and personal philosophies. It is imperative health care providers strive towards increased awareness and knowledge of how culture affects the overall experience of illness and death in order to help create a mutually satisfactory care plan. Providing culturally-competent care entails the use of proper communication skills to facilitate the exploration of patient and family perspectives and allows for mutual decision making. A case study will illustrate the challenges encountered in providing culturally-competent care to a woman with brain cancer and her family. As the patient's health declined, the family entered into a state of crisis where communication between family members and health care professionals was strained; leading to conflict and sub-optimal outcomes. This paper will address the ethical dilemma of providing culturally-competent care when a patient's safety is at risk, and the nursing implications of upholding best practices in the context of differing beliefs and priorities.

  19. Gene delivery into primary brain capillary endothelial cells for protein secretion

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek;

    model was established by co-culturing primary BCECs together with primary astrocytes, both of which were isolated from rats. This was done in order to study the possibility of using gene transfection in an environment closer to the in-vivo BBB situation. The in-vitro BBB barrier model showed trans...... for recombinant protein production. We have previously shown that it is possible to transfect human microvascular endothelial cells (HBMEC) and rat brain endothelial (RBE4) cells with genetic material encoding the human growth hormone (GH1) (Thomsen et al 2011). In the present study, however, an in-vitro BBB...

  20. Language evolution as cultural evolution: how language is shaped by the brain. (United States)

    Chater, Nick; Christiansen, Morten H


    This paper reviews arguments against the evolutionary plausibility of a traditional genetically specified universal grammar. We argue that no such universal grammar could have evolved, either by a process of natural selection or by other evolutionary mechanisms. Instead, we propose that the close fit between languages and language learners, which make language acquisition possible, arises not because humans possess a specialized biological adaptation for language, but because language has been shaped to fit the brain, a process of cultural evolution. On this account, many aspects of the structure of human languages may be explained as cultural adaptations to the human brain. Copyright © 2010 John Wiley & Sons, Ltd. For further resources related to this article, please visit the WIREs website.

  1. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)


    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  2. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  3. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation. (United States)

    Bozzone, Donna M.


    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  4. Brains, innovations, tools and cultural transmission in birds, non-human primates, and fossil hominins. (United States)

    Lefebvre, Louis


    Recent work on birds and non-human primates has shown that taxonomic differences in field measures of innovation, tool use and social learning are associated with size of the mammalian cortex and avian mesopallium and nidopallium, as well as ecological traits like colonization success. Here, I review this literature and suggest that many of its findings are relevant to hominin intelligence. In particular, our large brains and increased intelligence may be partly independent of our ape phylogeny and the result of convergent processes similar to those that have molded avian and platyrrhine intelligence. Tool use, innovativeness and cultural transmission might be linked over our past and in our brains as operations of domain-general intelligence. Finally, colonization of new areas may have accompanied increases in both brain size and innovativeness in hominins as they have in other mammals and in birds, potentially accelerating hominin evolution via behavioral drive.

  5. TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression. (United States)

    Simon, Melissa J; Gao, Shan; Kang, Woo Hyeun; Banta, Scott; Morrison, Barclay


    Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.

  6. Intravenous multipotent adult progenitor cell therapy after traumatic brain injury: modulation of the resident microglia population

    Directory of Open Access Journals (Sweden)

    Walker Peter A


    Full Text Available Abstract Introduction We have demonstrated previously that the intravenous delivery of multipotent adult progenitor cells (MAPC after traumatic brain injury affords neuroprotection via interaction with splenocytes, leading to an increase in systemic anti-inflammatory cytokines. We hypothesize that the observed modulation of the systemic inflammatory milieu is related to T regulatory cells and a subsequent increase in the locoregional neuroprotective M2 macrophage population. Methods C57B6 mice were injected with intravenous MAPC 2 and 24 hours after controlled cortical impact injury. Animals were euthanized 24, 48, 72, and 120 hours after injury. In vivo, the proportion of CD4+/CD25+/FOXP3+ T-regulatory cells were measured in the splenocyte population and plasma. In addition, the brain CD86+ M1 and CD206+ M2 macrophage populations were quantified. A series of in vitro co-cultures were completed to investigate the need for direct MAPC:splenocyte contact as well as the effect of MAPC therapy on M1 and M2 macrophage subtype apoptosis and proliferation. Results Significant increases in the splenocyte and plasma T regulatory cell populations were observed with MAPC therapy at 24 and 48 hours, respectively. In addition, MAPC therapy was associated with an increase in the brain M2/M1 macrophage ratio at 24, 48 and 120 hours after cortical injury. In vitro cultures of activated microglia with supernatant derived from MAPC:splenocyte co-cultures also demonstrated an increase in the M2/M1 ratio. The observed changes were secondary to an increase in M1 macrophage apoptosis. Conclusions The data show that the intravenous delivery of MAPC after cortical injury results in increases in T regulatory cells in splenocytes and plasma with a concordant increase in the locoregional M2/M1 macrophage ratio. Direct contact between the MAPC and splenocytes is required to modulate activated microglia, adding further evidence to the central role of the spleen in MAPC

  7. Patient-derived stem cells: pathways to drug discovery for brain diseases

    Directory of Open Access Journals (Sweden)

    Alan eMackay-Sim


    Full Text Available The concept of drug discovery through stem cell biology is based on technological developments whose genesis is now coincident. The first is automated cell microscopy with concurrent advances in image acquisition and analysis, known as high content screening (HCS. The second is patient-derived stem cells for modelling the cell biology of brain diseases. HCS has developed from the requirements of the pharmaceutical industry for high throughput assays to screen thousands of chemical compounds in the search for new drugs. HCS combines new fluorescent probes with automated microscopy and computational power to quantify the effects of compounds on cell functions. Stem cell biology has advanced greatly since the discovery of genetic reprogramming of somatic cells into induced pluripotent stem cells (iPSCs. There is now a rush of papers describing their generation from patients with various diseases of the nervous system. Although the majority of these have been genetic diseases, iPSCs have been generated from patients with complex diseases (schizophrenia and sporadic Parkinson’s disease. Some genetic diseases are also modelled in embryonic stem cells generated from blastocysts rejected during in vitro fertilisation. Neural stem cells have been isolated from post-mortem brain of Alzheimer’s patients and neural stem cells generated from biopsies of the olfactory organ of patients is another approach. These olfactory neurosphere-derived cells demonstrate robust disease-specific phenotypes in patients with schizophrenia and Parkinson’s disease. High content screening is already in use to find small molecules for the generation and differentiation of embryonic stem cells and induced pluripotent stem cells. The challenges for using stem cells for drug discovery are to develop robust stem cell culture methods that meet the rigorous requirements for repeatable, consistent quantities of defined cell types at the industrial scale necessary for high

  8. Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures. (United States)

    García-Heredia, José M; Hervás, Manuel; De la Rosa, Miguel A; Navarro, José A


    Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

  9. Human brain microvascular endothelial cells resist elongation due to shear stress. (United States)

    Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C


    Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier.

  10. Spatial organization and correlations of cell nuclei in brain tumors.

    Directory of Open Access Journals (Sweden)

    Yang Jiao

    Full Text Available Accepting the hypothesis that cancers are self-organizing, opportunistic systems, it is crucial to understand the collective behavior of cancer cells in their tumorous heterogeneous environment. In the present paper, we ask the following basic question: Is this self-organization of tumor evolution reflected in the manner in which malignant cells are spatially distributed in their heterogeneous environment? We employ a variety of nontrivial statistical microstructural descriptors that arise in the theory of heterogeneous media to characterize the spatial distributions of the nuclei of both benign brain white matter cells and brain glioma cells as obtained from histological images. These descriptors, which include the pair correlation function, structure factor and various nearest neighbor functions, quantify how pairs of cell nuclei are correlated in space in various ways. We map the centroids of the cell nuclei into point distributions to show that while commonly used local spatial statistics (e.g., cell areas and number of neighboring cells cannot clearly distinguish spatial correlations in distributions of normal and abnormal cell nuclei, their salient structural features are captured very well by the aforementioned microstructural descriptors. We show that the tumorous cells pack more densely than normal cells and exhibit stronger effective repulsions between any pair of cells. Moreover, we demonstrate that brain gliomas are organized in a collective way rather than randomly on intermediate and large length scales. The existence of nontrivial spatial correlations between the abnormal cells strongly supports the view that cancer is not an unorganized collection of malignant cells but rather a complex emergent integrated system.

  11. A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion. (United States)

    Sabra, Georges; Vermette, Patrick


    The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered.

  12. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells. (United States)

    Rieske, Piotr; Augelli, Brian J; Stawski, Robert; Gaughan, John; Azizi, S Ausim; Krynska, Barbara


    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

  13. Callus production from photoautotrophic soybean cell culture protoplasts. (United States)

    Chowhury, V K; Widholm, J M


    Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.

  14. HEPES inhibits the conversion of prion protein in cell culture. (United States)

    Delmouly, Karine; Belondrade, Maxime; Casanova, Danielle; Milhavet, Ollivier; Lehmann, Sylvain


    HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrP(Sc)). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

  15. Growth hormone (GH), brain development and neural stem cells. (United States)

    Waters, M J; Blackmore, D G


    A range of observations support a role for GH in development and function of the brain. These include altered brain structure in GH receptor null mice, and impaired cognition in GH deficient rodents and in a subgroup of GH receptor defective patients (Laron dwarfs). GH has been shown to alter neurogenesis, myelin synthesis and dendritic branching, and both the GH receptor and GH itself are expressed widely in the brain. We have found a population of neural stem cells which are activated by GH infusion, and which give rise to neurons in mice. These stem cells are activated by voluntary exercise in a GH-dependent manner. Given the findings that local synthesis of GH occurs in the hippocampus in response to a memory task, and that GH replacement improves memory and cognition in rodents and humans, these new observations warrant a reappraisal of the clinical importance of GH replacement in GH deficient states.

  16. Primary cell culture of human adenocarcinomas--practical considerations. (United States)

    Lerescu, Lucian; Tucureanu, Cătălin; Caraş, Iuliana; Neagu, Stefan; Melinceanu, Laura; Sălăgeanu, Aurora


    Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology. Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues. The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism. Unfortunately, such a model does not currently exist. Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells. Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories. The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data. Yet, their properties better reflect the cellular interactions present in intact tissue. The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject. The article also includes an updated protocol for primary tumor cell isolation and culture.


    Directory of Open Access Journals (Sweden)

    A.M. NOSOV


    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  18. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    Directory of Open Access Journals (Sweden)

    Samantha M. Grist


    Full Text Available The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications.

  19. Histamine from Brain Resident MAST Cells Promotes Wakefulness and Modulates Behavioral States


    Sachiko Chikahisa; Tohru Kodama; Atsushi Soya; Yohei Sagawa; Yuji Ishimaru; Hiroyoshi Séi; Seiji Nishino


    Mast cell activation and degranulation can result in the release of various chemical mediators, such as histamine and cytokines, which significantly affect sleep. Mast cells also exist in the central nervous system (CNS). Since up to 50% of histamine contents in the brain are from brain mast cells, mediators from brain mast cells may significantly influence sleep and other behaviors. In this study, we examined potential involvement of brain mast cells in sleep/wake regulations, focusing espec...

  20. The preventive effects of neural stem cells and mesenchymal stem cells intra-ventricular injection on brain stroke in rats

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    Seyed Mojtaba Hosseini


    Full Text Available Introduction: Stroke is one of the most important causes of disability in developed countries and, unfortunately, there is no effective treatment for this major problem of central nervous system (CNS; cell therapy may be helpful to recover this disease. In some conditions such as cardiac surgeries and neurosurgeries, there are some possibilities of happening brain stroke. Inflammation of CNS plays an important role in stroke pathogenesis, in addition, apoptosis and neural death could be the other reasons of poor neurological out come after stroke. In this study, we examined the preventive effects of the neural stem cells (NSCs and mesenchymal stem cells (MSCs intra-ventricular injected on stroke in rats. Aim: The aim of this study was to investigate the preventive effects of neural and MSCs for stroke in rats. Materials and Methods: The MSCs were isolated by flashing the femurs and tibias of the male rats with appropriate media. The NSCs were isolated from rat embryo ganglion eminence and they cultured NSCs media till the neurospheres formed. Both NSCs and MSCs were labeled with PKH26-GL. One day before stroke, the cells were injected into lateral ventricle stereotactically. Results: During following for 28 days, the neurological scores indicated that there are better recoveries in the groups received stem cells and they had less lesion volume in their brain measured by hematoxylin and eosin staining. Furthermore, the activities of caspase-3 were lower in the stem cell received groups than control group and the florescent microscopy images showed that the stem cells migrated to various zones of the brains. Conclusion: Both NSCs and MSCs are capable of protecting the CNS against ischemia and they may be good ways to prevent brain stroke consequences situations.

  1. Biologic characteristics of fibroblast cells cultured from the knee ligaments

    Institute of Scientific and Technical Information of China (English)

    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿


    Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

  2. LIF-free embryonic stem cell culture in simulated microgravity.

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    Yumi Kawahara

    Full Text Available BACKGROUND: Leukemia inhibitory factor (LIF is an indispensable factor for maintaining mouse embryonic stem (ES cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. CONCLUSIONS/SIGNIFICANCE: Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.

  3. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system. (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo


    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  4. Two sexually dimorphic cell groups in the human brain. (United States)

    Allen, L S; Hines, M; Shryne, J E; Gorski, R A


    A quantitative analysis of the volume of 4 cell groups in the preoptic-anterior hypothalamic area (PO-AHA) and of the supraoptic nucleus (SON) of the human brain was performed in 22 age-matched male and female individuals. We suggest the term Interstitial Nuclei of the Anterior Hypothalamus (INAH 1-4) to identify these 4 previously undescribed cell groups in the PO-AHA. While 2 INAH and the SON were not sexually dimorphic, gender-related differences were found in the other 2 cell groups. One nucleus (INAH-3) was 2.8 times larger in the male brain than in the female brain irrespective of age. The other cell group (INAH-2) was twice as large in the male brain, but also appeared to be related in women to circulating steroid hormone levels. Since the PO-AHA influences gonadotropin secretion, maternal behavior, and sexual behavior in several mammalian species, these results suggest that functional sex differences in the hypothalamus may be related to sex differences in neural structure.

  5. Nanoparticle accumulation and transcytosis in brain endothelial cell layers

    NARCIS (Netherlands)

    Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A


    The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight juncti

  6. Colorimetric pH measurement of animal cell culture media. (United States)

    Jang, Juno; Moon, Soo-Jin; Hong, Sung-Hwan; Kim, Ik-Hwan


    Most animal cell culture media can be buffered using bicarbonate and high pressure CO(2) in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO(2) pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.

  7. Drosophila neural stem cells in brain development and tumor formation. (United States)

    Jiang, Yanrui; Reichert, Heinrich


    Neuroblasts, the neural stem cells in Drosophila, generate the complex neural structure of the central nervous system. Significant progress has been made in understanding the mechanisms regulating the self-renewal, proliferation, and differentiation in Drosophila neuroblast lineages. Deregulation of these mechanisms can lead to severe developmental defects and the formation of malignant brain tumors. Here, the authors review the molecular genetics of Drosophila neuroblasts and discuss some recent advances in stem cell and cancer biology using this model system.

  8. [Application of cell co-culture techniques in medical studies]. (United States)

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


    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.

  9. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method. (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong


    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  10. Culture materials affect ex vivo expansion of hematopoietic progenitor cells. (United States)

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M


    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  11. Transplantation of autologous bone marrow-derived mesenchymal stem cells for traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    Jindou Jiang; Xingyao Bu; Meng Liu; Peixun Cheng


    Results from the present study demonstrated that transplantation of autologous bone marrow-derived mesenchymal stem cells into the lesion site in rat brain significantly ameliorated brain tissue pathological changes and brain edema, attenuated glial cell proliferation, and increased brain-derived neurotrophic factor expression. In addition, the number of cells double-labeled for 5-bromodeoxyuridine/glial fibrillary acidic protein and cells expressing nestin increased. Finally, blood vessels were newly generated, and the rats exhibited improved motor and cognitive functions. These results suggested that transplantation of autologous bone marrow-derived mesenchymal stem cells promoted brain remodeling and improved neurological functions following traumatic brain injury.

  12. Brain pericytes increase the lipopolysaccharide-enhanced transcytosis of HIV-1 free virus across the in vitro blood–brain barrier: evidence for cytokine-mediated pericyte-endothelial cell crosstalk (United States)


    Background Human immunodeficiency virus-1 (HIV-1) enters the brain by crossing the blood–brain barrier (BBB) as both free virus and within infected immune cells. Previous work showed that activation of the innate immune system with lipopolysaccharide (LPS) enhances free virus transport both in vivo and across monolayer monocultures of brain microvascular endothelial cells (BMECs) in vitro. Methods Here, we used monocultures and co-cultures of brain pericytes and brain endothelial cells to examine the crosstalk between these cell types in mediating the LPS-enhanced permeation of radioactively-labeled HIV-1 (I-HIV) across BMEC monolayers. Results We found that brain pericytes when co-cultured with BMEC monolayers magnified the LPS-enhanced transport of I-HIV without altering transendothelial electrical resistance, indicating that pericytes affected the transcytotic component of HIV-1 permeation. As LPS crosses the BBB poorly if at all, and since pericytes are on the abluminal side of the BBB, we postulated that luminal LPS acts indirectly on pericytes through abluminal secretions from BMECs. Consistent with this, we found that the pattern of secretion of cytokines by pericytes directly exposed to LPS was different than when the pericytes were exposed to the abluminal fluid from LPS-treated BMEC monolayers. Conclusion These results are evidence for a cellular crosstalk in which LPS acts at the luminal surface of the brain endothelial cell, inducing abluminal secretions that stimulate pericytes to release substances that enhance the permeability of the BMEC monolayer to HIV. PMID:23816186

  13. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture. (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales


    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  14. Three-dimensional cell culture models for investigating human viruses. (United States)

    He, Bing; Chen, Guomin; Zeng, Yi


    Three-dimensional (3D) culture models are physiologically relevant, as they provide reproducible results, experimental flexibility and can be adapted for high-throughput experiments. Moreover, these models bridge the gap between traditional two-dimensional (2D) monolayer cultures and animal models. 3D culture systems have significantly advanced basic cell science and tissue engineering, especially in the fields of cell biology and physiology, stem cell research, regenerative medicine, cancer research, drug discovery, and gene and protein expression studies. In addition, 3D models can provide unique insight into bacteriology, virology, parasitology and host-pathogen interactions. This review summarizes and analyzes recent progress in human virological research with 3D cell culture models. We discuss viral growth, replication, proliferation, infection, virus-host interactions and antiviral drugs in 3D culture models.

  15. LIF-Free Embryonic Stem Cell Culture in Simulated Microgravity


    Yumi Kawahara; Tomotaka Manabe; Masaya Matsumoto; Teruyuki Kajiume; Masayasu Matsumoto; Louis Yuge


    BACKGROUND: Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and...

  16. Exosomes function in cell-cell communication during brain circuit development


    Sharma, Pranav; Schiapparelli, Lucio; Cline, Hollis T.


    Exosomes are small extracellular vesicles that mediate intercellular signaling in the brain without requiring direct contact between cells. Although exosomes have been shown to play a role in neurological diseases and in response to nerve trauma, a role for exosome-mediated signaling in brain development and function has not yet been demonstrated. Here we review data building a case for exosome function in the brain.

  17. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype


    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.


    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

  18. Astrocyte Cultures Mimicking Brain Astrocytes in Gene Expression, Signaling, Metabolism and K(+) Uptake and Showing Astrocytic Gene Expression Overlooked by Immunohistochemistry and In Situ Hybridization. (United States)

    Hertz, Leif; Chen, Ye; Song, Dan


    Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K(+) concentrations and for Ca(2+) homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K(+) uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K(+) effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.

  19. Computational neuroanatomy: mapping cell-type densities in the mouse brain, simulations from the Allen Brain Atlas (United States)

    Grange, Pascal


    The Allen Brain Atlas of the adult mouse (ABA) consists of digitized expression profiles of thousands of genes in the mouse brain, co-registered to a common three-dimensional template (the Allen Reference Atlas).This brain-wide, genome-wide data set has triggered a renaissance in neuroanatomy. Its voxelized version (with cubic voxels of side 200 microns) is available for desktop computation in MATLAB. On the other hand, brain cells exhibit a great phenotypic diversity (in terms of size, shape and electrophysiological activity), which has inspired the names of some well-studied cell types, such as granule cells and medium spiny neurons. However, no exhaustive taxonomy of brain cell is available. A genetic classification of brain cells is being undertaken, and some cell types have been chraracterized by their transcriptome profiles. However, given a cell type characterized by its transcriptome, it is not clear where else in the brain similar cells can be found. The ABA can been used to solve this region-specificity problem in a data-driven way: rewriting the brain-wide expression profiles of all genes in the atlas as a sum of cell-type-specific transcriptome profiles is equivalent to solving a quadratic optimization problem at each voxel in the brain. However, the estimated brain-wide densities of 64 cell types published recently were based on one series of co-registered coronal in situ hybridization (ISH) images per gene, whereas the online ABA contains several image series per gene, including sagittal ones. In the presented work, we simulate the variability of cell-type densities in a Monte Carlo way by repeatedly drawing a random image series for each gene and solving the optimization problem. This yields error bars on the region-specificity of cell types.

  20. Marrow stromal cells administrated intracisternally to rats after traumatic brain injury migrate into the brain and improve neurological function

    Institute of Scientific and Technical Information of China (English)

    胡德志; 周良辅; 朱剑虹


    @@ Marrow stromal cells(MSCs) have been reported to transplant into injured brain via intravenous or intraarterial or direct intracerebral administration.1-3 In the present study, we observed that MSCs migrated into the brain, survived and diffeneriated into neural cells after they were injected into the cisterna magna of rats, and that the behavior of the rats after traumatic brain injury (TBI) was improved.

  1. The histopathology of Candida albicans invasion in neonatal rat tissues and in the human blood-brain barrier in culture revealed by light, scanning, transmission and immunoelectron microscopy scanning


    Lossinsky, A.S.; de Jong, A.; Fiala, M; Mukhtar, M; Buttle, K.F.; Ingram, M.


    The present studies examined the effects of Candida albicans yeast and hyphal morphologies on tissue pathologies and transmigration properties of the fungus in two experimental models: 1) an in vivo, neonatal rat model, and 2) a cell culture model of human brain microvascular endothelial cells (ECs) (BMVEC). We inoculated a hyphae-producing strain (CAI4-URA3) and a non-hyphae-producing strain (CAI4) of C. albicans into 4-10 day old rats and BMVEC cultures. ...

  2. Accessible cultural mind-set modulates default mode activity: evidence for the culturally situated brain. (United States)

    Wang, Chenbo; Oyserman, Daphna; Liu, Qiang; Li, Hong; Han, Shihui


    Self-construal priming modulates human behavior and associated neural activity. However, the neural activity associated with the self-construal priming procedure itself remains unknown. It is also unclear whether and how self-construal priming affects neural activity prior to engaging in a particular task. To address this gap, we scanned Chinese adults, using functional magnetic resonance imaging, during self-construal priming and a following resting state. We found that, relative to a calculation task, both interdependent and independent self-construal priming activated the ventral medial prefrontal cortex (MPFC) and the posterior cingulate cortex (PCC). The contrast of interdependent vs. independent self-construal priming also revealed increased activity in the dorsal MPFC and left middle frontal cortex. The regional homogeneity analysis of the resting-state activity revealed increased local synchronization of spontaneous activity in the dorsal MPFC but decreased local synchronization of spontaneous activity in the PCC when contrasting interdependent vs. independent self-construal priming. The functional connectivity analysis of the resting-state activity, however, did not show significant difference in synchronization of activities in remote brain regions between different priming conditions. Our findings suggest that accessible collectivistic/individualistic mind-set induced by self-construal priming is associated with modulations of both task-related and resting-state activity in the default mode network.

  3. Isolating highly pure rat spermatogonial stem cells in culture. (United States)

    Hamra, F Kent; Chapman, Karen M; Wu, Zhuoru; Garbers, David L


    Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cultured in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen when cultured in serum-containing medium. The collagen-non-binding testis cells obtained using these procedures are thus approx. 97% pure spermatogenic cells. Stem spermatogonia are then easily isolated from the purified spermatogenic population during a short incubation step in culture on laminin matrix. The spermatogenic cells that bind to laminin are more than 90% undifferentiated, type A spermatogonia and are greatly enriched in genetically modifiable stem cells that can develop into functional spermatozoa. This method does not require flow cytometry and can also be applied to obtain enriched cultures of mouse spermatogonial stem cells. The isolated spermatogonia provide a highly potent and effective source of stem cells that have been used to initiate in vitro and in vivo culture studies on spermatogenesis.

  4. Three Dimensional Culture of Human Renal Cell Carcinoma Organoids.

    Directory of Open Access Journals (Sweden)

    Cynthia A Batchelder

    Full Text Available Renal cell carcinomas arise from the nephron but are heterogeneous in disease biology, clinical behavior, prognosis, and response to systemic therapy. Development of patient-specific in vitro models that efficiently and faithfully reproduce the in vivo phenotype may provide a means to develop personalized therapies for this diverse carcinoma. Studies to maintain and model tumor phenotypes in vitro were conducted with emerging three-dimensional culture techniques and natural scaffolding materials. Human renal cell carcinomas were individually characterized by histology, immunohistochemistry, and quantitative PCR to establish the characteristics of each tumor. Isolated cells were cultured on renal extracellular matrix and compared to a novel polysaccharide scaffold to assess cell-scaffold interactions, development of organoids, and maintenance of gene expression signatures over time in culture. Renal cell carcinomas cultured on renal extracellular matrix repopulated tubules or vessel lumens in renal pyramids and medullary rays, but cells were not observed in glomeruli or outer cortical regions of the scaffold. In the polysaccharide scaffold, renal cell carcinomas formed aggregates that were loosely attached to the scaffold or free-floating within the matrix. Molecular analysis of cell-scaffold constructs including immunohistochemistry and quantitative PCR demonstrated that individual tumor phenotypes could be sustained for up to 21 days in culture on both scaffolds, and in comparison to outcomes in two-dimensional monolayer cultures. The use of three-dimensional scaffolds to engineer a personalized in vitro renal cell carcinoma model provides opportunities to advance understanding of this disease.

  5. Microfluidic cardiac cell culture model (μCCCM). (United States)

    Giridharan, Guruprasad A; Nguyen, Mai-Dung; Estrada, Rosendo; Parichehreh, Vahidreza; Hamid, Tariq; Ismahil, Mohamed Ameen; Prabhu, Sumanth D; Sethu, Palaniappan


    Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (μCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

  6. Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain (United States)

    Boulanger, Jenna J.; Messier, Claude


    Key Points Oligodendrocyte precursor cells express doublecortin, a microtubule-associated protein.Oligodendrocyte precursor cells express doublecortin, but at a lower level of expression than in neuronal precursor.Doublecortin is not associated with a potential immature neuronal phenotype in Oligodendrocyte precursor cells. Oligodendrocyte precursor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during embryogenesis and early stages of post-natal life. OPCs continue to divide throughout adulthood and some eventually differentiate into oligodendrocytes in response to demyelinating lesions. There is growing evidence that OPCs are also involved in activity-driven de novo myelination of previously unmyelinated axons and myelin remodeling in adulthood. Considering these roles in the adult brain, OPCs are likely mobile cells that can migrate on some distances before they differentiate into myelinating oligodendrocytes. A number of studies have noted that OPCs express doublecortin (DCX), a microtubule-associated protein expressed in neural precursor cells and in migrating immature neurons. Here we describe the distribution of DCX in OPCs. We found that almost all OPCs express DCX, but the level of expression appears to be much lower than what is found in neural precursor. We found that DCX is downregulated when OPCs start expressing mature oligodendrocyte markers and is absent in myelinating oligodendrocytes. DCX does not appear to signal an immature neuronal phenotype in OPCs in the adult mouse brain. Rather, it could be involved either in cell migration, or as a marker of an immature oligodendroglial cell phenotype.

  7. Development of bone marrow mesenchymal stem cell culture in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li; PENG Li-pan; WU Nan; LI Le-ping


    Objective To review the in vitro development of bone marrow mesenchymal stem cells culture (BM-MSC).Data sources The data cited in this review were mainly obtained from articles listed in Medline and PubMed.The search terms were “bone marrow mesenchymal stem cell" and "cell culture".Study selection Articles regarding the in vitro development of BM-MSCs culture,as well as the challenge of optimizing cell culture environment in two-dimensional (2D) vs.3D.Results Improving the culture conditions increases the proliferation and reduces the differentiation.Optimal values for many culture parameters remain to be identified.Expansion of BM-MSCs under defined conditions remains challenging,including the development of optimal culture conditions for BMSC and large-volume production systems.Conclusions Expansion of BM-MSCs under defined conditions remains challenges,including the development of optimal culture conditions for BMSC and scale-up to large-volume production systems.Optimal values for many culture parameters remain to be identified.

  8. Cell proliferation and cell death are disturbed during prenatal and postnatal brain development after uranium exposure. (United States)

    Legrand, M; Elie, C; Stefani, J; N Florès; Culeux, C; Delissen, O; Ibanez, C; Lestaevel, P; Eriksson, P; Dinocourt, C


    The developing brain is more susceptible to neurotoxic compounds than adult brain. It is also well known that disturbances during brain development cause neurological disorders in adulthood. The brain is known to be a target organ of uranium (U) exposure and previous studies have noted that internal U contamination of adult rats induces behavioral disorders as well as affects neurochemistry and neurophysiological properties. In this study, we investigated whether depleted uranium (DU) exposure affects neurogenesis during prenatal and postnatal brain development. We examined the structural morphology of the brain, cell death and finally cell proliferation in animals exposed to DU during gestation and lactation compared to control animals. Our results showed that DU decreases cell death in the cortical neuroepithelium of gestational day (GD) 13 embryos exposed at 40mg/L and 120mg/L and of GD18 fetuses exposed at 120mg/L without modification of the number of apoptotic cells. Cell proliferation analysis showed an increase of BrdU labeling in the dentate neuroepithelium of fetuses from GD18 at 120mg/L. Postnatally, cell death is increased in the dentate gyrus of postnatal day (PND) 0 and PND5 exposed pups at 120mg/L and is associated with an increase of apoptotic cell number only at PND5. Finally, a decrease in dividing cells is observed in the dentate gyrus of PND21 rats developmentally exposed to 120mg/L DU, but not at PND0 and PND5. These results show that DU exposure during brain development causes opposite effects on cell proliferation and cell death processes between prenatal and postnatal development mainly at the highest dose. Although these modifications do not have a major impact in brain morphology, they could affect the next steps of neurogenesis and thus might disrupt the fine organization of the neuronal network.

  9. Tomographic brain imaging with nucleolar detail and automatic cell counting (United States)

    Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schweighauser, Gabriel; Hench, Jürgen; Chicherova, Natalia; Schulz, Georg; Müller, Bert


    Brain tissue evaluation is essential for gaining in-depth insight into its diseases and disorders. Imaging the human brain in three dimensions has always been a challenge on the cell level. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Herein, we show that hard X-ray phase tomography can visualise a volume of up to 43 mm3 of human post mortem or biopsy brain samples, by demonstrating the method on the cerebellum. We automatically identified 5,000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, we highlight that three-dimensional data allows for the segmentation of sub-cellular structures, including dendritic tree and Purkinje cell nucleoli, without dedicated staining. The method suggests that automatic cell feature quantification of human tissues is feasible in phase tomograms obtained with isotropic resolution in a label-free manner.


    Institute of Scientific and Technical Information of China (English)


    There are many reports that cells (protoplasts) separated from the thallus of Porphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.

  11. Surface modified alginate microcapsules for 3D cell culture (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin


    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  12. Nylon-3 polymers that enable selective culture of endothelial cells. (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S


    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  13. Fundamentals of microfluidic cell culture in controlled microenvironments. (United States)

    Young, Edmond W K; Beebe, David J


    Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology.

  14. Police culture influences the brain function underlying compassion: a gender study. (United States)

    Mercadillo, Roberto E; Alcauter, Sarael; Fernández-Ruiz, Juan; Barrios, Fernando A


    Compassion is a prototypical moral emotion supporting cooperation and involves empathic decision-making and motor processes representing the interplay of biologically evolved and cultural mechanisms. We propose a social neuroscience approach to identify gender differences and to assess biological and cultural factors shaping compassion. We consider the police force as a cultural model to study this emotion, because it comprises a mixed-gender group using specific codes for collective safety that influence empathy and cooperativeness. From a sample of Mexican police officers working in a violent environment we integrated ethnographic data categorizing compassionate elements in the officers' activities, psychometric measures evaluating empathic attitudes, and fMRI scans identifying the brain activity related to compassionate experiences and decisions. The results suggest that the police culture influences genders equally with respect to empathic behavioral expressions. Nevertheless, women showed insular and prefrontal cortical activation, suggesting a more empathic experience of compassion. Officers manifested activity in the caudate nucleus, amygdala, and cerebellum, suggesting a more a highly accurate process to infer another's suffering and a reward system motivated by the notion of service and cooperation, both of which are cultural traits represented in the police force.

  15. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans


    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  16. Determining the Oxidation States of Manganese in NT2 Cells and Cultured Astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gunter,K.; Aschner, M.; Miller, L.; Eliseev, R.; Salter, J.; Andersen, K.; Gunter, T.


    Excessive brain manganese (Mn) can produce a syndrome called 'manganism', which correlates with loss of striatal dopamine and cell death in the striatum and globus pallidus. The prevalent hypothesis for the cause of this syndrome has been oxidation of cell components by the strong oxidizing agent, Mn{sup 3+}, either formed by oxidation of intracellular Mn{sup 2+} or transported into the cell as Mn{sup 3+}. We have recently used X-ray absorption near edge structure spectroscopy (XANES) to determine the oxidation states of manganese complexes in brain and liver mitochondria and in nerve growth factor (NGF)-induced and non-induced PC12 cells. No evidence was found for stabilization or accumulation of Mn{sup 3+} complexes because of oxidation of Mn{sup 2+} by reactive oxygen species in these tissues. Here we extend these studies of manganese oxidation state to cells of brain origin, human neuroteratocarcinoma (NT2) cells and primary cultures of rat astrocytes. Again we find no evidence for stabilization or accumulation of any Mn{sup 3+} complex derived from oxidation of Mn{sup 2+} under a range of conditions.

  17. Fucoidan Extracted from Hijiki Protects Brain Microvessel Endothelial Cells Against Diesel Exhaust Particle Exposure-Induced Disruption. (United States)

    Choi, Young-Sook; Eom, Sang-Yong; Kim, In-Soo; Ali, Syed F; Kleinman, Michael T; Kim, Yong-Dae; Kim, Heon


    This study was performed to evaluate the protective effects of fucoidan against the decreased function of primary cultured bovine brain microvessel endothelial cells (BBMECs) after exposure to diesel exhaust particles (DEPs). BBMECs were extracted from bovine brains and cultured until confluent. To evaluate the function of BBMECs, we performed a permeability test using cell-by-cell equipment and by Western blot analysis for zonular occludens-1 (ZO-1), which is a tight junction protein of BMECs, and evaluated oxidative stress in BBMECs using the DCFH-DA assay and the CUPRAC-BCS assay. The increased oxidative stress in BBMECs following DEP exposure was suppressed by fucoidan. In addition, permeability of BBMECs induced by DEP exposure was decreased by fucoidan treatment. Our results showed that fucoidan protects against BBMEC disruption induced by DEP exposure. This study provides evidence that fucoidan might protect the central nervous system (CNS) against DEP exposure.

  18. Sendai virus-based liposomes enable targeted cytosolic delivery of nanoparticles in brain tumor-derived cells

    Directory of Open Access Journals (Sweden)

    Dudu Veronica


    Full Text Available ABSTRACT Background Nanotechnology-based bioassays that detect the presence and/or absence of a combination of cell markers are increasingly used to identify stem or progenitor cells, assess cell heterogeneity, and evaluate tumor malignancy and/or chemoresistance. Delivery methods that enable nanoparticles to rapidly detect emerging, intracellular markers within cell clusters of biopsies will greatly aid in tumor characterization, analysis of functional state and development of treatment regimens. Results Experiments utilized the Sendai virus to achieve in vitro, cytosolic delivery of Quantum dots in cells cultured from Human brain tumors. Using fluorescence microscopy and Transmission Electron Microscopy, in vitro experiments illustrated that these virus-based liposomes decreased the amount of non-specifically endocytosed nanoparticles by 50% in the Human glioblastoma and medulloblastoma samples studied. Significantly, virus-based liposome delivery also facilitated targeted binding of Quantum dots to cytosolic Epidermal Growth Factor Receptor within cultured cells, focal to the early detection and characterization of malignant brain tumors. Conclusions These findings are the first to utilize the Sendai virus to achieve cytosolic, targeted intracellular binding of Qdots within Human brain tumor cells. The results are significant to the continued applicability of nanoparticles used for the molecular labeling of cancer cells to determine tumor heterogeneity, grade, and chemotherapeutic resistivity.

  19. Ontogeny of electrically excitable cells in cultured olfactory epithelium.


    Schubert, D; Stallcup, W.; LaCorbiere, M; Kidokoro, Y; Orgel, L


    A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the di...

  20. Characterization of a novel miniature cell culture device (United States)

    Moore, Sandra K.; Kleis, Stanley J.


    Recent advancements in the field of microfluidics have generated much interest in the advent of a miniaturized cell culture device. In this study, we developed a novel miniature culture system (cells, either prokaryotic or eukaryotic in type, for both 1 g and microgravity applications. The miniature culture system may advance the development of microanalytical remote monitoring tools such as biological sentinels, biosensors, and lab-on-a-chip. Integrating the autonomous miniature culture system with a microanalytical device makes a powerful biological tool. Cells can be cultured long-term, harvested, and released directly into an analytical tool without the need for human interaction through fluid dynamic manipulations. This work characterizes the miniature bioreactor system through numerical and experimental proof of concept studies.

  1. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G


    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  2. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

    Shah, Pranjul Jaykumar; Vedarethinam, Indumathi; Kwasny, Dorota


    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through...

  3. Transcranial amelioration of inflammation and cell death after brain injury (United States)

    Roth, Theodore L.; Nayak, Debasis; Atanasijevic, Tatjana; Koretsky, Alan P.; Latour, Lawrence L.; McGavern, Dorian B.


    Traumatic brain injury (TBI) is increasingly appreciated to be highly prevalent and deleterious to neurological function. At present, no effective treatment options are available, and little is known about the complex cellular response to TBI during its acute phase. To gain insights into TBI pathogenesis, we developed a novel murine closed-skull brain injury model that mirrors some pathological features associated with mild TBI in humans and used long-term intravital microscopy to study the dynamics of the injury response from its inception. Here we demonstrate that acute brain injury induces vascular damage, meningeal cell death, and the generation of reactive oxygen species (ROS) that ultimately breach the glial limitans and promote spread of the injury into the parenchyma. In response, the brain elicits a neuroprotective, purinergic-receptor-dependent inflammatory response characterized by meningeal neutrophil swarming and microglial reconstitution of the damaged glial limitans. We also show that the skull bone is permeable to small-molecular-weight compounds, and use this delivery route to modulate inflammation and therapeutically ameliorate brain injury through transcranial administration of the ROS scavenger, glutathione. Our results shed light on the acute cellular response to TBI and provide a means to locally deliver therapeutic compounds to the site of injury.

  4. PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture. (United States)

    Huang, Ruijie; Zhang, Junjie; Yang, X Frank; Gregory, Richard L


    Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.

  5. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. (United States)

    Lippmann, Ethan S; Azarin, Samira M; Kay, Jennifer E; Nessler, Randy A; Wilson, Hannah K; Al-Ahmad, Abraham; Palecek, Sean P; Shusta, Eric V


    The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover, because of its barrier properties, this endothelial interface restricts uptake of neurotherapeutics. Thus, a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues, including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes, including well-organized tight junctions, appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably, they respond to astrocytes, acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2), and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients.

  6. Application of cell co-culture system to study fat and muscle cells. (United States)

    Pandurangan, Muthuraman; Hwang, Inho


    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  7. Modulation of cellular adhesion in bovine brain microvessel endothelial cells by a decapeptide. (United States)

    Pal, D; Audus, K L; Siahaan, T J


    The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood-brain barrier (BBB) is poorly understood. Both Ca(2+)-dependent and Ca(2+)-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca(2+)-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-gamma or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH2, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca2+ were able to reaggregate only in the presence of Ca2+. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca(2+)-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.

  8. Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance. (United States)

    Li, Jincai; Wong, Chun Loong; Vijayasankaran, Natarajan; Hudson, Terry; Amanullah, Ashraf


    Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.

  9. Expression of precerebellins in cultured rat calvaria osteoblast-like cells. (United States)

    Rucinski, Marcin; Zok, Agnieszka; Guidolin, Diego; De Caro, Raffaele; Malendowicz, Ludwik K


    Cerebellin (CER), originally isolated from rat cerebellum, is a hexadecapeptide derived from the larger precursor called precerebellin 1 (Cbln1). At present 4 propeptides designated as Cbln1, Cbln2, Cbln3 and Cbln4 are recognized. They belong to precerebellin subfamily of the C1q family proteins. Precerebellins act as transneuronal regulators of synapse development and synaptic plasticity in various brain regions. Initially CER was thought to be a cerebellum specific peptide, however subsequent studies revealed its presence in other brain regions as well as in extraneuronal tissues. We investigated whether precerebellins are expressed and involved in regulation of cultured rat calvarial osteoblast-like (ROB) cells. Classic RT-PCR revealed the presence of Cbln1 and Cbln3 mRNA in fragments of rat calvaria, in freshly isolated ROB cells and in ROB cells cultured for 7, 14 and 21 days. Cbln2 and Cbln4 mRNA, on the other hand, could not be demonstrated in ROB cells but was found to be present in the brain. In freshly isolated ROB cells expression of Cbln1 gene was very low and gradually increased in relation to the duration of culture. Expression of Cbln3, on the other hand, was very low in fragments of rat calvaria, and increased notably after digestion with collagenase-I. The highest expression of this precerebellin was observed at day 14 of culture while at days 7 and 21 levels of expressions were notably lower. Neither Cbln2 nor Cbln4 was found to be expressed in the ROB cells. Neither CER nor des-Ser1-CER (10(-10)-10(-6)M) affect osteocalcin production and proliferation rate of studied cells. The above findings suggest that CER, which theoretically would be derived from Cbln1, modulate neither differentiated (osteocalcin secretion) nor basic (proliferation) functions of cultured rat osteoblast-like cells. The obtained data raise an intriguing hypothesis that precerebellins may be involved in regulating of spatial organization of osteoblastic niches in the bone.

  10. Adherence of Moraxella bovis to cell cultures of bovine origin. (United States)

    Annuar, B O; Wilcox, G E


    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  11. Cell/Tissue Culture Radiation Exposure Facility Project (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  12. Culture of graft-infiltrating cells from cryopreserved endomyocardial biopsies

    NARCIS (Netherlands)

    G.A. Patijn (G.); L.M.B. Vaessen (Leonard); W. Weimar (Willem); F.H.J. Claas (Frans); N.H.P.M. Jutte (Nicolet)


    textabstractGraft-infiltrating cells can be cultured from fresh endomyocardial biopsies (EMB) taken after heart transplantation to determine their growth patterns, phenotypic composition, and functional characteristics for clinical or scientific purposes. In this study we investigated whether graft-

  13. MicroRNAs in neural cell development and brain diseases. (United States)

    Feng, Wei; Feng, Yue


    MicroRNAs play important roles in post-transcriptional regulation of gene expression by inhibiting protein translation and/or promoting mRNA degradation. Importantly, biogenesis of microRNAs displays specific temporal and spatial profiles in distinct cell and tissue types and hence affects a broad spectrum of biological functions in normal cell growth and tumor development. Recent discoveries have revealed sophisticated mechanisms that control microRNA production and homeostasis in response to developmental and extracellular signals. Moreover, a link between dysregulation of microRNAs and human brain disorders has become increasingly evident. In this review, we focus on recent advances in understanding the regulation of microRNA biogenesis and function in neuronal and glial development in the mammalian brain, and dysregulation of the microRNA pathway in neurodevelopmental and neurodegenerative diseases.

  14. Which form of collagen is suitable for nerve cell culture?*

    Institute of Scientific and Technical Information of China (English)

    Mohsen Fathi Najafi; Saber Zahri; Fatemeh Vahedi; Leila Esmaililian Toosi; Nazila Ariaee


    In this study, we investigated the effects of hydrolyzed and non-hydrolyzed col agen and two-dimensional and three-dimensional col agen matrices on cell survival, attachment and neurite outgrowth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and inverted microscopy. Hydrolyzed col agen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cellattachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed col agen is an ideal nerve cell culture media.

  15. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun'ai; WANG Jingang; FAN Jinping; GONG Shufang; CHE Daidi


    Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg-L-1.When transfered onto subculture media,fi-iable callus developed into embryogenic callus,which was used to establish cell suspension lines.Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles.The best subculturing cycle for the stable cell suspensions was 8-10 days.The best inoculum quantity was 1 mL PCV(Packed Cell Volume) per 40 mL culture fluid.

  16. 2D- and 3D-culture of cell

    Directory of Open Access Journals (Sweden)

    Khoruzhenko A. I.


    Full Text Available The cultivation of mammalian cells in three-dimensional conditions acquires a priority in a variety of biomedical applications. In the areas of toxicology and anticancer drug development it concerns a significant difference of responses to proapoptotic factors of the cells cultured in 2D versus 3D environment. Besides, the clear-cut differences have been found in cell polarity, cytoskeleton structure, distribution of receptors to wide range of hormones, growth factors, etc. in mammalian cells depending on culture conditions. It is resulted in different response of cultured cells to extracellular stimuli. Multicellular spheroids are regarded presently as the most convenient model of solid tumour growth in vitro. The cultivation of thyroid follicles, mammary acini and other structure units, maintaining initial tissue organization, allows studying the behavior, biochemical features and gene profile of differentiated cells. On the other hand, 3D cultures have some limitations in comparison with a well established monolayer culture. The advantages and disadvantages of each type of cultures and their application in biological and medical researches will be discussed in this review

  17. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;


    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long...

  18. Antioxidative effects of Panax notoginseng saponins in brain cells. (United States)

    Zhou, Ningna; Tang, Yang; Keep, Richard F; Ma, Xiaoxia; Xiang, Jianming


    Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death associated with neurological disorders such as stroke, Alzheimer's disease and traumatic brain injury. Antioxidant compounds that improve endogenous antioxidant defenses have been proposed for neural protection. The purpose of this study was to investigate the potential protective effects of total saponin in leaves of Panax notoginseng (LPNS) on oxidative stress and cell death in brain cells in vitro. Lactate dehydrogenase (LDH) assay indicated that LPNS (5 μg/ml) reduced H2O2-induced cell death in primary rat cortical astrocytes (23±8% reduction in LDH release vs. control). Similar protection was found in oxygen and glucose deprivation/reoxygenation induced SH-SY5Y (a human neuroblastoma cell line) cell damage (78±7% reduction vs. control). The protective effects of LPNS in astrocytes were associated with attenuation of reactive oxygen species (ROS) accumulation. These effects involved activation of Nrf2 (nuclear translocation) and upregulation of downstream antioxidant systems including heme oxygenase-1 (HO-1) and glutathione S-transferase pi 1 (GSTP1). These results demonstrate for the first time that LPNS has antioxidative effects which may be neuroprotective in neurological disorders.

  19. Convoluted cells as a marker for maternal cell contamination in CVS cultures

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Jensen, P K; Therkelsen, A J


    In order to identify cells of maternal origin in CVS cultures, tissue from 1st trimester abortions were cultivated and the cultures stained in situ for X-chromatin. Convoluted cells and maternal fibroblasts were found to be positive. By chromosome analysis of cultures from 105 diagnostic placenta...... biopsies, obtained by the transabdominal route, metaphases of maternal origin were found in nine cases. In eight of these cases colonies of convoluted cells were observed. We conclude that convoluted cells are of maternal origin and are a reliable marker for maternal cell contamination in CVS cultures....

  20. Guard cell protoplasts: isolation, culture, and regeneration of plants. (United States)

    Tallman, Gary


    Guard cell protoplasts have been used extensively in short-term experiments designed to elucidate the signal transduction mechanisms that regulate stomatal movements. The utility of uard cell protoplasts for other types of longer-term signal transduction experiments is just now being realized. Because highly purified, primary isolates of guard cell protoplasts are synchronous initially, they are uniform in their responses to changes in culture conditions. Such isolates have demonstrated potential to reveal mechanisms that underlie hormonal signalling for plant cell survival, cell cycle re-entry, reprogramming of genes during dedifferentiation to an embryogenic state, and plant cell thermotolerance. Plants have been regenerated from cultured guard cell protoplasts of two species: Nicotiana glauca (Graham), tree tobacco, and Beta vulgaris, sugar beet. Plants genetically engineered for herbicide tolerance have been regenerated from cultured guard cell protoplasts of B. vulgaris. The method for isolating, culturing, and regenerating plants from guard cell protoplasts of N. glauca is described here. A recently developed procedure for large-scale isolation of these cells from as many as nine leaves per experiment is described. Using this protocol, yields of 1.5-2 x 10(7) per isolate may be obtained. Such yields are sufficient for standard methods of molecular, biochemical, and proteomic analysis.

  1. Xeno-free culture of human periodontal ligament stem cells. (United States)

    Trubiani, Oriana; Diomede, Francesca


    The possibility of transplanting adult stem cells into damaged organs has opened a new prospective for the treatment of several human pathologies. Currently, in vitro expansion and culture of mesenchymal stem cells is founded on supplementing cell culture and differentiation medium with fetal calf serum (FCS) or fetal bovine serum (FBS) that contain numerous growth factors inducing cell attachment to plastic surfaces, proliferation, and differentiation. Mesenchymal stem cells (MSCs) cultured with medium containing FCS or FBS are unusable in the cell therapy; in fact the central issues regarding limitations in using animal sera for cell therapy is that its components are highly variable and often unknown and may trigger a xenogenic immune response, immunological reactions, and the potential transmission of prion diseases and zoonoses. Here we describe the culture system protocols for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free medium formulation ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy.

  2. Auxin requirements of sycamore cells in suspension culture. (United States)

    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C


    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  3. Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Filipa L Cardoso

    Full Text Available BACKGROUND: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS is known to alter the integrity of the blood-brain barrier (BBB, little is known on the effects of unconjugated bilirubin (UCB and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC. METHODOLOGY/PRINCIPAL FINDINGS: Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. CONCLUSIONS: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.

  4. Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle. (United States)

    Musarò, Antonio; Carosio, Silvia


    Skeletal muscle tissue is characterized by a population of quiescent mononucleated myoblasts, localized between the basal lamina and sarcolemma of myofibers, known as satellite cells. Satellite cells play a pivotal role in muscle homeostasis and are the major source of myogenic precursors in mammalian muscle regeneration.This chapter describes protocols for isolation and culturing satellite cells isolated from mouse skeletal muscles. The classical procedure, which will be discussed extensively in this chapter, involves the enzymatic dissociation of skeletal muscles, while the alternative method involves isolation of satellite cells from isolated myofibers in which the satellite cells remain in their in situ position underneath the myofiber basal lamina.In particular, we discuss the technical aspect of satellite cell isolation, the methods necessary to enrich the satellite cell fraction and the culture conditions that optimize proliferation and myotube formation of mouse satellite cells.

  5. Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders

    Directory of Open Access Journals (Sweden)

    Natalie Kaminsky


    Full Text Available The DNA damage response (DDR is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes. Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a “hostile” environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.

  6. Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders

    Institute of Scientific and Technical Information of China (English)

    Natalie Kaminsky; Ofer Bihari; Sivan Kanner; Ari Barzilai


    The DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to geno-mic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degener-ative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evi-dence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes). Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a‘‘hostile”environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.

  7. The permeation of dynorphin A 1-6 across the blood brain barrier and its effect on bovine brain microvessel endothelial cell monolayer permeability. (United States)

    Sloan, Courtney D Kuhnline; Audus, Kenneth L; Aldrich, Jane V; Lunte, Susan M


    Dynorphin A 1-17 (Dyn A 1-17) is an endogenous neuropeptide known to act at the kappa opioid receptor; it has been implicated in a number of neurological disorders, including neuropathic pain, stress, depression, and Alzheimer's and Parkinson's diseases. The investigation of Dyn A 1-17 metabolism at the blood-brain barrier (BBB) is important since the metabolites exhibit unique biological functions compared to the parent compound. In this work, Dyn A 1-6 is identified as a metabolite of Dyn A 1-17 in the presence of bovine brain microvessel endhothelial cells (BBMECs), using LC-MS/MS. The transport of Dyn A 1-6 at the BBB was examined using this in vitro cell culture model of the BBB. Furthermore, the permeation of the BBB by the low molecular weight permeability marker fluorescein was characterized in the presence and absences of Dyn A 1-6.

  8. [Effect evaluation of three cell culture models]. (United States)

    Wang, Aiguo; Xia, Tao; Yuan, Jing; Chen, Xuemin


    Primary rat hepatocytes were cultured using three kinds of models in vitro and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH in the medium decreased over time in the period of culture. However, on 5 days, LDH showed a significant increase in monolayer culture (MC) while after 8 days LDH was not detected in sandwich culture (SC). The levels of AST and ALT in the medium did not change significantly over the investigated time. The basic CYP 1A activity gradually decreased with time in MC and SC. The decline of CYP 1A in rat hepatocytes was faster in MC than that in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducers such as omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than that in SC. Basic CYP 1A activity in bioreactor was keeped over 2 weeks and the highest albumin production was observed in bioreactor, and next were SC and MC. In conclusion, our results clearly indicated that there have some advantages and disadvantages in each of models in which can address different questions in metabolism of toxicants and drugs.

  9. Qualitative study of three cell culture methods. (United States)

    Wang, Aiguo; Xia, Tao; Ran, Peng; Chen, Xuemin; Nuessler, Andreas K


    Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.

  10. Lacrimal gland primary acinar cell culture: the role of insulin

    Directory of Open Access Journals (Sweden)

    Leonardo Tannus Malki


    Full Text Available ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL. Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

  11. Microtransplantation of membranes from cultured cells to Xenopus oocytes: A method to study neurotransmitter receptors embedded in native lipids


    Palma, Eleonora; Trettel, Flavia; Fucile, Sergio; Renzi, Massimiliano; Miledi, Ricardo; Eusebi, Fabrizio


    The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cel...

  12. Ephrin-A3 and ephrin-A4 contribute to microglia-induced angiogenesis in brain endothelial cells. (United States)

    Li, Ying; Liu, Dong-Xin; Li, Mei-Yang; Qin, Xiao-Xue; Fang, Wen-Gang; Zhao, Wei-Dong; Chen, Yu-Hua


    The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia-deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary-like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin-A3 and ephrin-A4, were specifically upregulated in BMECs exposed to BV2-derived culture supernatant. Knockdown of ephrin-A3 and ephrin-A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF-α) released from BV2 microglial cells. Moreover, the upregulations of ephrin-A3 and ephrin-A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF-α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin-A3 and ephrin-A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia-released TNF-α.

  13. From blood to brain: amoeboid microglial cell, a nascent macrophage and its functions in developing brain

    Institute of Scientific and Technical Information of China (English)

    Charanjit KAUR; S Thameem DHEEN; Eng-ang LING


    Amoeboid microglial cells (AMC) in the developing brain are active macrophages.The macrophagic nature of these cells has been demonstrated by many methods,such as the localization of various hydrolytic enzymes and the presence of comple-ment type 3 surface receptors in them. More importantly is the direct visualization of these cells engaged in the phagocytosis of degenerating cells at the ultrastruc-tural level. Further evidence of them being active macrophages is the avid inter-nalization of tracers administered by the intravenous or intraperitoneal routes in developing rats. The potential involvement of AMC in immune functions is sup-ported by the induced expression of major histocompatibility complex class Ⅰ and Ⅱ antigens on them when challenged by lipopolysaccharide or interferon-γ. Im-munosuppressive drugs, such as glucocorticoids and immune function-enhanc-ing drugs like melatonin, affect the expression of surface receptors and antigens and the release of cytokines by AMC. Recent studies in our laboratory have shown the expression of insulin-like growth factors, endothelins, 21,31-cyclic nucle-otide 31-phosphodiesterase, and N-methyl-D-asparate receptors. This along with the release of chemokines, such as stromal derived factor-la and monocyte chemoattractant protein-1, suggests multiple functional roles of AMC in early brain development.

  14. Morphological and protein profile comparison of large vessel and microvascular endothelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Beer, D.M.; Kim, J.S.; Carson, M.P.; Haudeuschild, C.C.; Patton, W.F.; Jacobson, B.S.


    Bovine adrenal medulla (AmMEC) and brain (BrMEC) microvessel endothelial cells, and bovine aortic (BAE) endothelial cells were isolated and cultured under identical conditions using a modification of a technique previously described for BrMEC. The cells were isolated and passaged under conditions minimizing cell surface alterations. Primary cultures were confluent in 4-6 days at a plating density in the region of 10/sup 4/ cells/cm/sup 2/. BAEs maintained a cobblestone morphology and a denser monolayer than MECs in primary and passaged cells whether the cells were passaged using Pancreatin, Trypsin-EDTA, or Collagenase-EDTA. MECs were initially elongate and became more like BAEs with passaging. BAEs and AmMECs were examined for differences in whole cell, Triton extracted cytoskeleton and plasma membrane (PM) protein profiles by two-dimensional gel electrophoresis. Cells were labeled with /sup 35/S-methionine and PM by lactoperoxidase catalyzed iodination. Though for the most part protein patterns were similar, several proteins in the PM and cytoskeletal preparations differed. A significant difference in the isoelectric forms of proteins with the same molecular weight was observed in the PM.

  15. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard


    Full Text Available Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  16. Hydrofocusing Bioreactor for Three-Dimensional Cell Culture (United States)

    Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly


    The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

  17. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan;


    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...

  18. Brown but not white adipose cells synthesize omega-3 docosahexaenoic acid in culture. (United States)

    Qin, Xia; Park, Hui Gyu; Zhang, Ji Yao; Lawrence, Peter; Liu, Guowen; Subramanian, Nivetha; Kothapalli, Kumar S D; Brenna, J Thomas


    Adipose tissue is a complex endocrine organ which coordinates several crucial biological functions including fatty acid metabolism, glucose metabolism, energy homeostasis, and immune function. Brown adipose tissue (BAT) is most abundant in young infants during the brain growth spurt when demands for omega-3 docosahexaenoic acid (DHA, 22:6n-3) is greatest for brain structure. Our aim was to characterize relative biosynthesis of omega-3 long chain polyunsaturated fatty acids (LCPUFA) from precursors in cultured white (WAT) and brown (BAT) cells and study relevant gene expression. Mouse WAT and BAT cells were grown in regular DMEM media to confluence, and differentiation was induced. At days 0 and 8 cells were treated with albumin bound d5-18:3n-3 (d5-ALA) and analyzed 24h later. d5-ALA increased cellular eicosapentaenoic acid (EPA, 20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) in undifferentiated BAT cells, whereas differentiated BAT cells accumulated 20:4n-3, EPA and DPA. DHA as a fraction of total omega-3 LCPUFA was greatest in differentiated BAT cells compared to undifferentiated cells. Undifferentiated WAT cells accumulated EPA, whereas differentiated cells accumulated DPA. WAT accumulated trace newly synthesized DHA. Zic1 a classical brown marker and Prdm16 a key driver of brown fat cell fate are expressed only in BAT cells. Ppargc1a is 15 fold higher in differentiated BAT cells. We conclude that in differentiated adipose cells accumulating fat, BAT cells but not WAT cells synthesize DHA, supporting the hypothesis that BAT is a net producer of DHA.

  19. Increased leakage of brain antigens after traumatic brain injury and effect of immune tolerance induced by cells on traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    YAN Hua; ZHANG Hong-wei; WU Qiao-li; ZHANG Guo-bin; LIU Kui; ZHI Da-shi; HU Zhen-bo; ZENG Xian-wei


    Background Although traumatic brain injury can lead to opening the blood-brain barrier and leaking of blood substances (including water) into brain tissue,few studies of brain antigens leaking into the blood and the pathways have been reported.Brain antigens result in damage to brain tissues by stimulating the immune system to produce anti-brain antibodies,but no treatment has been reported to reduce the production of anti-brain antibodies and protect the brain tissue.The aim of the study is to confirm the relationship between immune injury and arachnoid granulations following traumatic brain injury,and provide some new methods to inhibit the immune injury.Methods In part one,methylene blue was injected into the rabbits' cisterna magna after traumatic brain injury,and concentrations of methylene blue and tumor necrosis factor (TNF)-α in blood were detected to determine the permeability of arachnoid granulations.In part two,umbilical cord mesenchymal stem cells and immature dendritic cells were injected into veins,and concentrations of interleukin 1 (IL-1),IL-10,interferon (IFN)-y,transforming growth factor (TGF)-β,anti-brain antibodies (ABAb),and IL-12 were measured by ELISA on days 1,3,7,14 and 21 after injury,and the numbers of leukocytes in the blood were counted.Twenty-one days after injury,expression of glutamate in brain tissue was determined by immunohistochemical staining,and neuronal degeneration was detected by H&E staining.Results In part one,blood concentrations of methylene blue and TNF-α in the traumatic brain injury group were higher than in the control group (P <0.05).Concentrations of methylene blue and TNF-α in the trauma cerebrospinal fluid (CSF)injected group were higher than in the control cerebrospinal fluid injected group (P <0.05).In part two,concentrations of IL-1,IFN-y,ABAb,IL-12,expression of glutamate (Glu),neuronal degeneration and number of peripheral blood leukocytes were lower in the group with cell treatment compared to the

  20. Good Cell Culture Practice for stem cells and stem-cell-derived models. (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas


    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  1. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.


    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...


    NARCIS (Netherlands)



    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of e

  3. Learning about Cells as Dynamic Entities: An Inquiry-Driven Cell Culture Project (United States)

    Palombi, Peggy Shadduck; Jagger, Kathleen Snell


    Using cultured fibroblast cells, undergraduate students explore cell division and the responses of cultured cells to a variety of environmental changes. The students learn new research techniques and carry out a self-designed experiment. Through this project, students enhance their creative approach to scientific inquiry, learn time-management and…

  4. Modeling of cell culture damage and recovery leads to increased antibody and biomass productivity in CHO cell cultures. (United States)

    Naderi, Saeideh; Nikdel, Ali; Meshram, Mukesh; McConkey, Brendan; Ingalls, Brian; Budman, Hector; Scharer, Jeno


    The development of an efficient and productive cell-culture process requires a deep understanding of intracellular mechanisms and extracellular conditions for optimal product synthesis. Mathematical modeling provides an effective strategy to predict, control, and optimize cell performance under a range of culture conditions. In this study, a mathematical model is proposed for the investigation of cell damage of a Chinese hamster ovary cell culture secreting recombinant anti-RhD monoclonal antibody (mAb). Irreversible cell damage was found to be correlated with a reduction in pH. This irreversible damage to cellular function is described mathematically by a Tessier-based model, in which the actively growing fraction of cells is dependent on an intracellular metabolic product acting as a growth inhibitor. To further verify the model, an offline model-based optimization of mAb production in the cell culture was carried out, with the goal of minimizing cell damage and thereby enhancing productivity through intermittent refreshment of the culture medium. An experimental implementation of this model-based strategy resulted in a doubling of the yield as compared to the batch operation and the resulting biomass and productivity profiles agreed with the model predictions.

  5. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage. (United States)

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki


    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  6. Nonlinear Dynamic Theory of Acute Cell Injuries and Brain Ischemia (United States)

    Taha, Doaa; Anggraini, Fika; Degracia, Donald; Huang, Zhi-Feng


    Cerebral ischemia in the form of stroke and cardiac arrest brain damage affect over 1 million people per year in the USA alone. In spite of close to 200 clinical trials and decades of research, there are no treatments to stop post-ischemic neuron death. We have argued that a major weakness of current brain ischemia research is lack of a deductive theoretical framework of acute cell injury to guide empirical studies. A previously published autonomous model based on the concept of nonlinear dynamic network was shown to capture important facets of cell injury, linking the concept of therapeutic to bistable dynamics. Here we present an improved, non-autonomous formulation of the nonlinear dynamic model of cell injury that allows multiple acute injuries over time, thereby allowing simulations of both therapeutic treatment and preconditioning. Our results are connected to the experimental data of gene expression and proteomics of neuron cells. Importantly, this new model may be construed as a novel approach to pharmacodynamics of acute cell injury. The model makes explicit that any pro-survival therapy is always a form of sub-lethal injury. This insight is expected to widely influence treatment of acute injury conditions that have defied successful treatment to date. This work is supported by NIH NINDS (NS081347) and Wayne State University President's Research Enhancement Award.

  7. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model (United States)

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  8. Nitric oxide consumption through lipid peroxidation in brain cell suspensions and homogenates. (United States)

    Keynes, Robert G; Griffiths, Charmaine H; Hall, Catherine; Garthwaite, John


    Mechanisms which inactivate NO (nitric oxide) are probably important in governing the physiological and pathological effects of this ubiquitous signalling molecule. Cells isolated from the cerebellum, a brain region rich in the NO signalling pathway, consume NO avidly. This property was preserved in brain homogenates and required both particulate and supernatant fractions. A purified fraction of the particulate component was rich in phospholipids, and NO consumption was inhibited by procedures that inhibited lipid peroxidation, namely a transition metal chelator, the vitamin E analogue Trolox and ascorbate oxidase. The requirement for the supernatant was accounted for by its content of ascorbate which catalyses metal-dependent lipid peroxidation. The NO-degrading activity of the homogenate was mimicked by a representative mixture of brain lipids together with ascorbate and, under these conditions, the lipids underwent peroxidation. In a suspension of cerebellar cells, there was a continuous low level of lipid peroxidation, and consumption of NO by the cells was decreased by approx. 50% by lipid-peroxidation inhibitors. Lipid peroxidation was also abolished when NO was supplied at a continuously low rate (approximately 100 nM/min), which explains why NO consumption by this process is saturable. Part of the activity remaining after the inhibition of lipid peroxidation was accounted for by contaminating red blood cells, but there was also another component whose activity was greatly enhanced when the cells were maintained under air-equilibrated conditions. A similar NO-consuming process was present in cerebellar glial cells grown in tissue culture but not in blood platelets or leucocytes, suggesting a specialized mechanism.

  9. Interactions of primary neuroepithelial progenitor and brain endothelial cells: distinct effect on neural progenitor maintenance and differentiation by soluble factors and direct contact

    Institute of Scientific and Technical Information of China (English)

    Miguel A Gama Sosa; Rita De Gasperi; Anne B Rocher; Gissel M Perez; Keila Simons; Daniel E Cruz; Patrick R Hof; Gregory A Elder


    Neurovascular interactions are crucial for the normal development of the central nervous system. To study such interactions in primary cultures, we developed a procedure to simultaneously isolate neural progenitor and endothelial cell fractions from embryonic mouse brains. Depending on the culture conditions endothelial cells were found to favor maintenance of the neuroprogenitor phenotype through the production of soluble factors, or to promote neuronal differentiation of neural progenitors through direct contact. These apparently opposing effects could reflect differential cellular interactions needed for the proper development of the brain.

  10. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen


    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of gro......, but all attempts to induce formation of shoots or em-bryoids gave negative results....

  11. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    Directory of Open Access Journals (Sweden)

    Efstathia Papafragkou

    Full Text Available Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407 or human epithelial colorectal adenocarcinoma cells (Caco-2 growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin. Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8. At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  12. Metabolism Kinetics of Glucose in Anchorage-dependent Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    孙祥明; 张元兴


    The kinetic model of glucose metabolism was established and successfully applied to batchcultures of rCHO and rBHK cells. It was found that a large amount of glucose was utilized for cellmaintenance, and the overwhelming majority of maintenance energy from glucose was by its anaerobicmetabolism in both rBHK and rCHO cell cultures. The overall maintenance coefficients from aerobicmetabolism were 1.9×10-13 mmol/(cell.h) for rCHO cells and 7×10-13 mmol/(cell.h) for rBHK cells. Inaddition, all Go/T and Eo/T gradually increased with the same trend as the cell growth in the culture ofboth rCHO and rBHK cells. The overall molecule yield coefficients of lactate to glucose were 1.61 for rCHO cells and 1.38 for rBHK cells. The yield coefficients of cell to glucose were 4.5×108 cells/mmol for rCHO cells and 1.9 × 108 cells/mmol for rBHK cells, respectively.

  13. Advances in culture and manipulation of human pluripotent stem cells. (United States)

    Qian, X; Villa-Diaz, L G; Krebsbach, P H


    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.

  14. In vitro methods to culture primary human breast epithelial cells. (United States)

    Raouf, Afshin; Sun, Yu Jia


    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  15. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture. (United States)

    Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz


    Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

  16. Establishment of 9L/F344 rat intracerebral glioma model of brain tumor stem cells

    Directory of Open Access Journals (Sweden)

    Zong-yu XIAO


    Full Text Available Objective To establish the 9L/F344 rat intracerebral glioma model of brain tumor stem cells.  Methods Rat 9L gliosarcoma stem-like cells were cultured in serum-free suspension. The expression of CD133 and nestin were tested by immunohistochemistry. A total of 48 inbredline male F344 rats were randomly divided into 2 groups, and 9L tumor sphere cells and 9L monolayer cells were respectively implanted into the right caudate nucleus of F344 rats in 2 groups. Survival time was observed and determined using the method of Kaplan-Meier survival analysis. Fourteen days after implantation or when the rats were dying, their brains were perfused and sectioned for HE staining, and CD133 and nestin were detected by immunohistochemistry.  Results Rat 9L tumor spheres were formed with suspension culture in serum-free medium. The gliomas formed in both groups were invasive without obvious capsule. More new vessels, bleeding and necrosis could be detected in 9L tumor spheres group. The tumor cells in both groups were positive for CD133 and nestin. There was no significant difference in the expression of CD133 and nestin between 2 groups (P > 0.05, for all. According to the expression of nestin, the tumors formed by 9L tumor sphere cells were more invasive. The median survival time of the rats bearing 9L tumor sphere cells was 15 d (95%CI: 15.219-15.781, and the median survival time of the rats bearing 9L monolayer cells was 21 d (95%CI: 20.395-21.605. There was significant difference between 2 groups (χ2 = 12.800, P = 0.000.  Conclusions 9L/F344 rat intracerebral glioma model of brain tumor stem cells is successfully established, which provides a glioma model for the future research. DOI: 10.3969/j.issn.1672-6731.2015.04.012

  17. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.


    Nagy, A.; Rossant, J.; Nagy, R.; Abramow-Newerly, W; Roder, J C


    Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and...

  18. Seed train optimization for cell culture. (United States)

    Frahm, Björn


    For the production of biopharmaceuticals a seed train is required to generate an adequate number of cells for inoculation of the production bioreactor. This seed train is time- and cost-intensive but offers potential for optimization. A method and a protocol are described for the seed train mapping, directed modeling without major effort, and its optimization regarding selected optimization criteria such as optimal points in time for cell passaging. Furthermore, the method can also be applied for the set-up of a new seed train, for example for a new cell line. Although the chapter is directed towards suspension cell lines, the method is also generally applicable, e.g. for adherent cell lines.

  19. Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

    Indian Academy of Sciences (India)

    Axel Blau; Tanja Neumann; Christiane Ziegler; Fabio Benfenati


    An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity overtime. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

  20. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate. (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I


    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  1. Cell cultures from the symbiotic soft coral Sinularia flexibilis

    NARCIS (Netherlands)

    Khalesi, M.K.; Vera-Jimenez, N.I.; Aanen, D.K.; Beeftink, H.H.; Wijffels, R.H.


    The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cu

  2. Isolation and Characterization of Poliovirus in Cell Culture Systems. (United States)

    Thorley, Bruce R; Roberts, Jason A


    The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

  3. Quantitative phase imaging for cell culture quality control. (United States)

    Kastl, Lena; Isbach, Michael; Dirksen, Dieter; Schnekenburger, Jürgen; Kemper, Björn


    The potential of quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for quantification of cell culture quality was explored. Label-free QPI of detached single cells in suspension was performed by Michelson interferometer-based self-interference DHM. Two pancreatic tumor cell lines were chosen as cellular model and analyzed for refractive index, volume, and dry mass under varying culture conditions. Firstly, adequate cell numbers for reliable statistics were identified. Then, to characterize the performance and reproducibility of the method, we compared results from independently repeated measurements and quantified the cellular response to osmolality changes of the cell culture medium. Finally, it was demonstrated that the evaluation of QPI images allows the extraction of absolute cell parameters which are related to cell layer confluence states. In summary, the results show that QPI enables label-free imaging cytometry, which provides novel complementary integral biophysical data sets for sophisticated quantification of cell culture quality with minimized sample preparation. © 2017 International Society for Advancement of Cytometry.

  4. Animal-cell culture in aqueous two-phase systems.

    NARCIS (Netherlands)

    Zijlstra, G.M.


    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in this the

  5. Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

    Institute of Scientific and Technical Information of China (English)

    Lirong Jin; Zhen Hong; Chunjiu Zhong; Yang Wang


    BACKGROUND: To date, the use of bone marrow-derived mesenchymal stem cells (MSCs) for the treatment of Parkinson's disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE: To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion (MPP+), and to investigate the effect of MSCs on dopaminergic neurons following MPP+ induced damage.DESIGN, TIME AND SETTING: An in vitro, randomized, controlled, animal experiment using immunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006.MATERIALS: Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase (TH, 1:5 000) were from Santa Cruz (USA); goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company (China).METHODS: Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode's balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups: control, MPP+ exposure, and co-culture. For the MPP+ group, MPP+ (30 μmol/L) was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP+ injured brain slices were placed over MSCs in the well and were further cultured for 7 days.MAIN OUTCOME MEASURES: After 28 days in culture, neurite outgrowth was examined in the brain slices under phase

  6. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia. (United States)

    Belzer, Vitali; Shraer, Nathanael; Hanani, Menachem


    Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.

  7. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

    Yaping Feng; Hui Zhu; Jiang Hao; Xinmin Wang; Shengping Wu; Li Bai; Xiangming Li; Yun Zha


    BACKGROUND:Previous studies have used many methods for in vitro Schwann cells (SCs) cul-tures and purification,such as single cell suspension and cytosine arabinoside.However,it has been difficult to obtain sufficient cellular density,and the procedures have been quite tedious.OBJECTIVE:To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants.DESIGN,TIME AND SETTING:Cell culture and immunohistochemistry were performed at the Cen-tral Laboratory of Kunming General Hospital of Chinese PLA between March 2001 and October 2008.MATERIALS:Culture media containing 10% fetal bovine serum,as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco,USA;mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Bi-ological Products,China.METHODS:Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fe-tuses at 4-6 months pregnancy.Following removal of the dorsal root ganglion perineurium,the gan-glia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1),then explanted and cultured.SC purification was performed with 5 mL 10% fetal bovine serum added to the culture media,followed by differential adhesion.MAIN OUTCOME MEASURES:SCs morphology was observed under inverted phase contrast light microscopy.SC purity was evaluated according to percentage of S-100 immunostained cells.RESULTS:SCs were primarily cultured for 5-6 days and then subcultured for 4-5 passages.The highly enriched SC population reached > 95% purity and presented with normal morphology.CONCLUSION:A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

  8. Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers


    Tamura Makoto; Tamura Naohiro; Ikeda Takamitsu; Koyama Ryuta; Ikegaya Yuji; Matsuki Norio; Yamada Maki K


    Abstract Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced a...

  9. Serpins promote cancer cell survival and vascular co-option in brain metastasis. (United States)

    Valiente, Manuel; Obenauf, Anna C; Jin, Xin; Chen, Qing; Zhang, Xiang H-F; Lee, Derek J; Chaft, Jamie E; Kris, Mark G; Huse, Jason T; Brogi, Edi; Massagué, Joan


    Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

  10. Miniature Bioreactor System for Long-Term Cell Culture (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.


    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  11. Cloning higher plants from aseptically cultured tissues and cells (United States)

    Krikorian, A. D.


    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  12. Radiosensitivity of cultured insect cells: I. Lepidoptera

    Energy Technology Data Exchange (ETDEWEB)

    Koval, T.M.


    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D/sub 0/, d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D/sub 0/ of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects.

  13. U251胶质瘤细胞系中脑肿瘤干细胞的分离、培养及鉴定%Isolation,Culture and Identification of Brain Tumor Stem Cellswithin U251 Glioma Cell Line In Vitro

    Institute of Scientific and Technical Information of China (English)

    陈成; 芦明; 李茗初; 方加胜


      Objective This study was to isolate, culture and identificate brain tumor stem cel s from U251 Gliome cel line in vitro and observe their growth pattern.To establish a fundation for further reseach of brain tumor stem cel . Methods U251cel s were seeded in serum-free DMEM-F12 medium supplemented with B27, EGF and bFGF in 6-wel plates, after U251 brain tumor spheres formed, they were dissociated and passaged in fresh medium periodical y. U251 brain tumor spheres and the single cel s of them were induced to differentiate in the medium with 10%FBS supplemented. U251cel s cultivate in serum-supplemented medium and serum-free medium alternately. At last we performed immunocytochemistry of U251 brain tumor spheres for CD133 and Nestin. Results Some U251 glioma cel have the capacity to self-renew, proliferate and generate free-floating neurosphere-like brain tumor spheres in serum-free medium in vitro. U251 glioma spheres and the single cel s of them can be induced to differentiate and adherent to the bottom of the culture plates. The growing patterns of U251 glioma cel line can be converted by changing the culture mediums. The brain tumor stem cel are CD133+Nestin+cel s. Conclusion The U251 glioma cel line contain brain tumor stem cel s which can be isolated, proliferated and differentiated in vitro. The research of brain tumor stem cel s may provide a new platform for brain tumor study.%  目的本研究旨在从U251胶质瘤细胞系中分离、培养和鉴定脑肿瘤干细胞,观察其的生长特征,为脑肿瘤干细胞的进一步研究打下基础。方法应用简化的无血清培养基和悬浮培养法培养U251细胞,并将获得的脑肿瘤干细胞接种于含血清培养基中观察其分化;交替用含血清培基和无血清培基培养U251细胞,观察其生长特性;用免疫荧光法检测脑肿瘤干细胞中CD133和Nestin的表达。结果 U251细胞接种至无血清培养基后,部分细胞能够存活并增殖为悬浮

  14. Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations. (United States)

    Naill, Michael C; Roberts, Susan C


    Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

  15. Cell culture monitoring for drug screening and cancer research: a transparent, microfluidic, multi-sensor microsystem. (United States)

    Weltin, Andreas; Slotwinski, Kinga; Kieninger, Jochen; Moser, Isabella; Jobst, Gerhard; Wego, Marcus; Ehret, Ralf; Urban, Gerald A


    We present a novel, multiparametric microphysiometry system for the dynamic online monitoring of human cancer cell metabolism. The optically transparent, modular, hybrid microsystem is based on a glass chip and combines a cell cultivation chamber, microfluidics and metabolic monitoring with fully integrated chemo- and biosensors. pH and oxygen are measured in the cell culture area, and biosensors for lactate and glucose are connected downstream by microfluidics. The wafer-level fabrication features thin-film platinum and iridium oxide microelectrodes on a glass chip, microfluidics in an epoxy resist, a hybrid assembly and an on-chip reference electrode. The reliable analytical performance of the sensors in cell culture medium was demonstrated. The pH sensors exhibit a long-term stable, linear response. The oxygen sensors show a linear behaviour, which is also observed for low oxygen concentrations. Glucose and lactate measurements show a linear, long-term stable, selective and reversible behaviour in the desired range. T98G human brain cancer cells were cultivated and cell culture metabolism was measured on-chip. Stop/flow cycles were applied and extracellular acidification, respiration, glucose consumption and lactate production were quantified. Long-term metabolic rates were determined and all parameters could be measured in the outlet channel. A placement downstream of the cell cultivation area for biosensors was realised. A highly effective medium exchange and undiluted sampling from the cell culture chamber with low flow rates (2 μl min(-1)) and low volumes (15 μl per cycle) were achieved. The drug screening application was demonstrated by detecting alteration and recovery effects of cellular metabolism induced by the addition of substances to the medium.

  16. Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean. (United States)

    Gamborg, O L; Davis, B P; Stahlhut, R W


    Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.

  17. Furan fatty acids efficiently rescue brain cells from cell death induced by oxidative stress

    NARCIS (Netherlands)

    Teixeira, A.; Cox, R.C.; Egmond, M.R.


    Treatment of rat brain C6 astroglioma cells with furan fatty acid F6 prior to exposure to hydrogen peroxide shows a strong protective effect of F6 against cell death resulting from oxidative stress. This protective effect is obtained only for F6 administered as a free fatty acid and with an intact f

  18. New findings about iron oxide nanoparticles and their different effects on murine primary brain cells. (United States)

    Neubert, Jenni; Wagner, Susanne; Kiwit, Jürgen; Bräuer, Anja U; Glumm, Jana


    The physicochemical properties of superparamagnetic iron oxide nanoparticles (SPIOs) enable their application in the diagnostics and therapy of central nervous system diseases. However, since crucial information regarding side effects of particle-cell interactions within the central nervous system is still lacking, we investigated the influence of novel very small iron oxide particles or the clinically approved ferucarbotran or ferumoxytol on the vitality and morphology of brain cells. We exposed primary cell cultures of microglia and hippocampal neurons, as well as neuron-glia cocultures to varying concentrations of SPIOs for 6 and/or 24 hours, respectively. Here, we show that SPIO accumulation by microglia and subsequent morphological alterations strongly depend on the respective nanoparticle type. Microglial viability was severely compromised by high SPIO concentrations, except in the case of ferumoxytol. While ferumoxytol did not cause immediate microglial death, it induced severe morphological alterations and increased degeneration of primary neurons. Additionally, primary neurons clearly degenerated after very small iron oxide particle and ferucarbotran exposure. In neuron-glia cocultures, SPIOs rather stimulated the outgrowth of neuronal processes in a concentration- and particle-dependent manner. We conclude that the influence of SPIOs on brain cells not only depends on the particle type but also on the physiological system they are applied to.

  19. [Neuroscience and collective memory: memory schemas linking brain, societies and cultures]. (United States)

    Legrand, Nicolas; Gagnepain, Pierre; Peschanski, Denis; Eustache, Francis


    During the last two decades, the effect of intersubjective relationships on cognition has been an emerging topic in cognitive neurosciences leading through a so-called "social turn" to the formation of new domains integrating society and cultures to this research area. Such inquiry has been recently extended to collective memory studies. Collective memory refers to shared representations that are constitutive of the identity of a group and distributed among all its members connected by a common history. After briefly describing those evolutions in the study of human brain and behaviors, we review recent researches that have brought together cognitive psychology, neuroscience and social sciences into collective memory studies. Using the reemerging concept of memory schema, we propose a theoretical framework allowing to account for collective memories formation with a specific focus on the encoding process of historical events. We suggest that (1) if the concept of schema has been mainly used to describe rather passive framework of knowledge, such structure may also be implied in more active fashions in the understanding of significant collective events. And, (2) if some schema researches have restricted themselves to the individual level of inquiry, we describe a strong coherence between memory and cultural frameworks. Integrating the neural basis and properties of memory schema to collective memory studies may pave the way toward a better understanding of the reciprocal interaction between individual memories and cultural resources such as media or education.

  20. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes. (United States)

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki


    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  1. Specific accumulation of {sup 18}F-deoxyglucose in three-dimensional long-term cultures of human and rodent brain tissue

    Energy Technology Data Exchange (ETDEWEB)

    Hocke, C.; Prante, O.; Kuwert, T. [Clinic of Nuclear Medicine, Univ. of Erlangen-Nuernberg (Germany); Bluemcke, I.; Jeske, I. [Dept. of Neuropathology, Univ. of Erlangen-Nuernberg (Germany); Romstoeck, J. [Dept. of Neurosurgery, Univ. of Erlangen-Nuernberg (Germany); Stefan, H. [Dept. of Neurology, Univ. of Erlangen-Nuernberg (Germany)


    Aim: Organotypic slice cultures (OSC) of human brain specimens represent an intriguing experimental model for translational studies addressing, e.g., stem cell transplantation in neurodegenerative diseases or targeting invasion by malignant glioma ex vivo. However, long-term viability and phenomena of structural reorganization of human OSC remain to be further characterized. Here, we report the use of {sup 18}F-deoxyglucose (FDG) for evaluating the viability of brain slice preparations obtained either from postnatal rats or human hippocampal specimens. Methods: Anatomically well preserved human hippocampi obtained from epilepsy surgery and rat hippocampus slice cultures obtained from six day old Wistar rats were dissected into horizontal slices. The slices were incubated with FDG in phosphate buffered saline up to 1 h, either with or without supplementation of glucose at a concentration of 2.5 mg/ml. Radioactivity within the medium or slice cultures was measured using a gamma-counter. In addition, distribution of radioactivity was autoradiographically visualized and quantified as counts per mm{sup 2}. Results: In rat hippocampal slices, FDG accumulated with 1 300 000 {+-} 68 000 counts/mm{sup 2}, whereas the incorporation of the radioactive label in human slices was in the order of 1 500 000 {+-} 370 000 counts/mm{sup 2}. The elevation of glucose concentration within the medium led to a significant three-fold decrease of FDG accumulation in rat slices and to a 2.4-fold decrease in human specimens. Conclusions: FDG accumulated in organotypic brain cultures of human or rodent origin. FDG is thus suited to investigate the viability of OSC. Furthermore, these preparations open new ways to study the factors governing cerebral FDG uptake in brain tissue ex vivo. (orig.)

  2. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. (United States)

    Krampe, Britta; Al-Rubeai, Mohamed


    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

  3. The replacement of serum by hormones in cell culture media. (United States)

    Sato, G; Hayashi, I


    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  4. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fulford, D.E.


    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  5. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture. (United States)

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide


    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45(-)Ter119(-)Dlk1(+) LPCs derived from murine foetal livers formed ALBUMIN (ALB)(+)CYTOKERATIN (CK)19(-) non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB(-)CK19(+) cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo.

  6. Neural activity control of neural stem cells and SVZ niche response to brain injury


    Páez González, Patricia


    Patricia Paez-Gonzalez Kuo Lab, Dept. of Cell Biology, Duke University Medical Center, NC,USA. Date: 11/16/2014 Utilizing stem cells in the adult brain hold great promise for regenerative medicine. Harnessing ability of adult neural stem cells (NSCs) to generate new neurons or other types of brain cells may provide much needed therapies for patients suffering from brain injuries or neuro-degenerative diseases such as Parkinson’s, Scizophrenia, or Alzheimer’s disease. However...

  7. Wave characterization for mammalian cell culture: residence time distribution. (United States)

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário


    The high dose requirements of biopharmaceutical products led to the development of mammalian cell culture technologies that increase biomanufacturing capacity. The disposable Wave bioreactor is one of the most promising technologies, providing ease of operation and no cross-contamination, and using an innovative undulation movement that ensures good mixing and oxygen transfer without cell damage. However, its recentness demands further characterization. This study evaluated the residence time distribution (RTD) in Wave, allowing the characterization of mixing and flow and the comparison with ideal models and a Stirred tank reactor (STR) used for mammalian cell culture. RTD was determined using methylene blue with pulse input methodology, at three flow rates common in mammalian cell culture (3.3×10(-5)m(3)/h, 7.9×10(-5)m(3)/h, and 1.25×10(-4)m(3)/h) and one typical of microbial culture (5×10(-3)m(3)/h). Samples were taken periodically and the absorbance read at 660nm. It was observed that Wave behavior diverted from ideal models, but was similar to STR. Therefore, the deviations are not related to the particular Wave rocking mechanism, but could be associated with the inadequacy of these reactors to operate in continuous mode or to a possible inability of the theoretical models to properly describe the behavior of reactors designed for mammalian cell culture. Thus, the development of new theoretical models could better characterize the performance of these reactors.

  8. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth


    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.


    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  9. Culture of Neural Stem Cells in Calcium-alginate Microbeads

    Institute of Scientific and Technical Information of China (English)

    Li-Song YAO; Tian-Qing LIU; Dan GE; Xue-Hu MA; Zhan-Feng CUI


    @@ 1 Introduction Recent research shows that neural stem cells may play an important role in the nerve injury reparation and nerve disease treatment. The shortage of the source and the number of NSCs, however, is the main challenge for its clinic application. In this situation, expansion of NSCs in large scale and culture in three dimensional environment are very worth of exploration. Notablely, the shear stress existed in bioreactors can cause serious cell injury especially for the shear sensitive cells like NSCs.

  10. Experiments on tissue culture in the genus Lycopersicon miller : Shoot formation from protoplasts of tomato long-term cell cultures. (United States)

    Koblitz, H; Koblitz, D


    Callus cultures from cotyledon explants were established and maintained in culture for more than two years. After several months callus cultures were transferred into liquid medium and cultured as cell suspensions. Protoplasts were isolated from these cell suspension cultures and cultured in a liquid medium. After formation of new cell walls the cells were further cultured in liquid medium and afterwards transferred to an agar-solidified medium to give a vigorously growing callus culture. In the case of the cultivar 'Lukullus' shoots were recovered from callus. All efforts to root these shoots failed and this, in addition to variations in appearence, suggests that the shoots are changed genetically possibly due to the prolonged culture period.

  11. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro. (United States)

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo


    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  12. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M


    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...

  13. A novel closed cell culture device for fabrication of corneal epithelial cell sheets. (United States)

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu


    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  14. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment

    Institute of Scientific and Technical Information of China (English)

    Xianchao Li; Wensheng Hou; Xiaoying Wu; Wei Jiang; Haiyan Chen; Nong Xiao; Ping Zhou


    Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hy-poxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efifciencies are relatively low. Red or near-infrared light from 600-1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migra-tion and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2, an increasing number of green lfuorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 × 106 bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2 for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental ifndings indicate that 660 nm red light emitting diode irradiation promotes the migration of bone marrow mesenchymal stem cells, thereby enhancing the contribution of cell transplantation in the treatment of hypox-ic-ischemic brain damage.

  15. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment. (United States)

    Li, Xianchao; Hou, Wensheng; Wu, Xiaoying; Jiang, Wei; Chen, Haiyan; Xiao, Nong; Zhou, Ping


    Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hypoxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efficiencies are relatively low. Red or near-infrared light from 600-1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migration and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cm(2), an increasing number of green fluorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 × 10(6) bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm(2) for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental findings indicate that 660 nm red light emitting diode irradiation promotes the migration of bone marrow mesenchymal stem cells, thereby enhancing the contribution of cell transplantation in the treatment of hypoxic-ischemic brain damage.

  16. Metabolic measurements in cell culture and tissue constructs (United States)

    Rolfe, P.


    This paper concerns the study and use of biological cells in which there is a need for sensors and assemblies for the measurement of a diverse range of physical and chemical variables. In this field cell culture is used for basic research and for applications such as protein and drug synthesis, and in cell, tissue and organ engineering. Metabolic processes are fundamental to cell behaviour and must therefore be monitored reliably. Basic metabolic studies measure the transport of oxygen, glucose, carbon dioxide, lactic acid to, from, or within cells, whilst more advanced research requires examination of energy storage and utilisation. Assemblies are designed to incorporate bioreactor functions for cell culture together with appropriate sensing devices. Oxygen consumption by populations of cells is achieved in a flowthrough assembly that incorporates O2 micro-sensors based on either amperometry or fluorescence. Measurements in single cell are possible with intra-cellular fluorophores acting as biosensors together with optical stimulation and detection. Near infra-red spectroscopy (NIRS) is used for analysis within culture fluid, for example for estimation of glucose levels, as well as within cell populations, for example to study the respiratory enzymes.Â#

  17. Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture. (United States)

    Laydi, F; Rahouadj, R; Cauchois, G; Stoltz, J-F; de Isla, N


    Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.

  18. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture. (United States)


    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed...

  19. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment. (United States)


    ... SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and equipment. (a) Identification. Cell and tissue culture... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and...

  20. Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2.

    Directory of Open Access Journals (Sweden)

    Julie Dwyer

    Full Text Available Glioblastoma constitutes the most aggressive and deadly of brain tumors. As yet, both conventional and molecular-based therapies have met with limited success in treatment of this cancer. Among other explanations, the heterogeneity of glioblastoma and the associated microenvironment contribute to its development, as well as resistance and recurrence in response to treatments. Increased vascularity suggests that tumor angiogenesis plays an important role in glioblastoma progression. However, the molecular crosstalk between endothelial and glioblastoma cells requires further investigation. To examine the effects of glioblastoma-derived signals on endothelial homeostasis, glioblastoma cell secretions were collected and used to treat brain endothelial cells. Here, we present evidence that the glioblastoma secretome provides pro-angiogenic signals sufficient to disrupt VE-cadherin-mediated cell-cell junctions and promote endothelial permeability in brain microvascular endothelial cells. An unbiased angiogenesis-specific antibody array screen identified the chemokine, interleukin-8, which was further demonstrated to function as a key factor involved in glioblastoma-induced permeability, mediated through its receptor CXCR2 on brain endothelia. This underappreciated interface between glioblastoma cells and associated endothelium may inspire the development of novel therapeutic strategies to induce tumor regression by preventing vascular permeability and inhibiting angiogenesis.

  1. Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity. (United States)

    Brajkovic, Saška; Ferdaoussi, Mourad; Pawlowski, Valérie; Ezanno, Hélène; Plaisance, Valérie; Zmuda, Erik; Hai, Tsonwin; Annicotte, Jean-Sébastien; Waeber, Gérard; Abderrahmani, Amar


    Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

  2. Growth enhancement effect of BzATP on primary cultured astrocytes from rat brain

    Institute of Scientific and Technical Information of China (English)

    Hua-Zheng LIANG; Ying LIU; Zhu-Rong YE


    Objective To explore whether BzATP could promote the growth of primary cultured astrocytes (AS) of rat and its possible mechanism, and whether TGF-β1 was involved in the event. Methods The primary cultured AS were derived from new born Sprague-Dawley rats.Glial fibrillary acidic protein (GFAP) immunofluorescent stain was used to check the purity of cultured AS. Morphometry was used to detect the changes of AS. The proliferation index of AS was detected by BrdU incorporation assay. Western blot was used to detect the changes of GFAP under different conditions. Changes of TGF-β1 gene transcription were detected by RT-PCR. ELISA was utilized to detect the variation of TGF-β1 protein in the supernate. Results The purity of primary cultured AS reached to 99%. BzATP promoted the hypertrophy of AS including the elongation of AS processes and the enlargement of cell bodies, BzATP also promoted the expression of GFAP in existence of Ca2+, but had no effect on cell proliferation. BzATP increased the transcription of TGF-β1 mRNA and the release of TGF-β1 protein in existence of Ca2+. TGF-β1 neutralizing antibody partially inhibited the expression of GFAP induced by BzATP, but had no effect on AS proliferation and cell morphology. Conclusion BzATP enhanced the hypertrophy of primary cultured AS, increased the expression of GFAP partially through TGF-β1. Mechanisms of the enhancement of AS growth induced by BzATP other than TGF-51 pathway remains to be elucidated.

  3. Hollow fiber clinostat for simulating microgravity in cell culture (United States)

    Rhodes, Percy H. (Inventor); Miller, Teresa Y. (Inventor); Snyder, Robert S. (Inventor)


    A clinostat for simulating microgravity on cell systems carried in a fiber fixedly mounted in a rotatable culture vessel is disclosed. The clinostat is rotated horizontally along its longitudinal axis to simulate microgravity or vertically as a control response. Cells are injected into the fiber and the ends of the fiber are sealed and secured to spaced end pieces of a fiber holder assembly which consists of the end pieces, a hollow fiber, a culture vessel, and a tension spring with three alignment pins. The tension spring is positioned around the culture vessel with its ends abutting the end pieces for alignment of the spring. After the fiber is secured, the spring is decompressed to maintain tension on the fiber while it is being rotated. This assures that the fiber remains aligned along the axis of rotation. The fiber assembly is placed in the culture vessel and culture medium is added. The culture vessel is then inserted into the rotatable portion of the clinostat and subjected to rotate at selected rpms. The internal diameter of the hollow fiber determines the distance the cells are from the axis of rotation.

  4. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  5. Experimental study of bioartificial liver with cultured human liver cells

    Institute of Scientific and Technical Information of China (English)


    AIM To establish an extracorporeal bioartificial liver support system (EBLSS) using cultured human liver cells and to study its support effect for fulminant hepatic failure (FHF).METHODS The liver support experiment of EBLSS consisting of aggregates cultured human liver cells, hollow fiber bioreactor, and circulation unit was carried out in dizhepatic dogs.RESULTS The viability of isolated hepatocytes and nonparenchymal liver cells reached 96%. These cells were successfully cultured as multicellular spheroids with synthetic technique. The typical morphological appearance was retained up to the end of the artificial liver experiment. Compared with the control dogs treated with EBLSS without liver cells, the survival time of artificial liver support dogs was significantly prolonged. The changes of blood pressure, heart rate and ECG were slow. Both serum ammonia and lactate levels were significantly lowered at the 3rd h and 5th h. In addition, a good viability of human liver cells was noted after 5 h experiment.CONCLUSION EBLSS playing a metabolic role of cultured human hepatocytes, is capable of compensating the function of the liver, and could provide effective artificial liver support and therapy for patients with FHF.

  6. [In vitro cell culture technology in cosmetology research]. (United States)

    Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata


    For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

  7. "Humanized" stem cell culture techniques: the animal serum controversy. (United States)

    Tekkatte, Chandana; Gunasingh, Gency Ponrose; Cherian, K M; Sankaranarayanan, Kavitha


    Cellular therapy is reaching a pinnacle with an understanding of the potential of human mesenchymal stem cells (hMSCs) to regenerate damaged tissue in the body. The limited numbers of these hMSCs in currently identified sources, like bone marrow, adipose tissue, and so forth, bring forth the need for their in vitro culture/expansion. However, the extensive usage of supplements containing xenogeneic components in the expansion-media might pose a risk to the post-transplantation safety of patients. This warrants the necessity to identify and develop chemically defined or "humanized" supplements which would make in vitro cultured/processed cells relatively safer for transplantation in regenerative medicine. In this paper, we outline the various caveats associated with conventionally used supplements of xenogenic origin and also portray the possible alternatives/additives which could one day herald the dawn of a new era in the translation of in vitro cultured cells to therapeutic interventions.

  8. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.


    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed...... and introducing the observed difference between batches as one of the factors in an expanded three-dimensional ANOVA, we were able to overcome an otherwisecrucial lack of sufficiently reproducible duplicate values. We could thereby show that the effect of changing the apical medium was much more marked when...... the radioactive lipid precursors were added on the apical, rather than on the basolateral, side. Theinsert cell cultures were obviously polarized. We argue that it is not reasonable to reject troublesome experimental results, when we do not know a priori that something went wrong. The ANOVA is a very useful...

  9. Beauty and the brain: culture, history and individual differences in aesthetic appreciation. (United States)

    Jacobsen, Thomas


    Human aesthetic processing entails the sensation-based evaluation of an entity with respect to concepts like beauty, harmony or well-formedness. Aesthetic appreciation has many determinants ranging from evolutionary, anatomical or physiological constraints to influences of culture, history and individual differences. There are a vast number of dynamically configured neural networks underlying these multifaceted processes of aesthetic appreciation. In the current challenge of successfully bridging art and science, aesthetics and neuroanatomy, the neuro-cognitive psychology of aesthetics can approach this complex topic using a framework that postulates several perspectives, which are not mutually exclusive. In this empirical approach, objective physiological data from event-related brain potentials and functional magnetic resonance imaging are combined with subjective, individual self-reports.

  10. Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

    Directory of Open Access Journals (Sweden)

    Quan Jiang


    Full Text Available Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury, substantially improve functional outcome. We discuss and review state of the art magnetic resonance imaging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

  11. Magnetic resonance imaging and cell-based neurorestorative therapy after brain injury

    Institute of Scientific and Technical Information of China (English)

    Quan Jiang


    Restorative cell-based therapies for experimental brain injury, such as stroke and traumatic brain injury, substantially improve functional outcome. We discuss and review state of the art magnetic resonance im-aging methodologies and their applications related to cell-based treatment after brain injury. We focus on the potential of magnetic resonance imaging technique and its associated challenges to obtain useful new information related to cell migration, distribution, and quantitation, as well as vascular and neuronal remodeling in response to cell-based therapy after brain injury. The noninvasive nature of imaging might more readily help with translation of cell-based therapy from the laboratory to the clinic.

  12. A novel feeder-free culture system for human pluripotent stem cell culture and induced pluripotent stem cell derivation.

    Directory of Open Access Journals (Sweden)

    Sanna Vuoristo

    Full Text Available Correct interactions with extracellular matrix are essential to human pluripotent stem cells (hPSC to maintain their pluripotent self-renewal capacity during in vitro culture. hPSCs secrete laminin 511/521, one of the most important functional basement membrane components, and they can be maintained on human laminin 511 and 521 in defined culture conditions. However, large-scale production of purified or recombinant laminin 511 and 521 is difficult and expensive. Here we have tested whether a commonly available human choriocarcinoma cell line, JAR, which produces high quantities of laminins, supports the growth of undifferentiated hPSCs. We were able to maintain several human pluripotent stem cell lines on decellularized matrix produced by JAR cells using a defined culture medium. The JAR matrix also supported targeted differentiation of the cells into neuronal and hepatic directions. Importantly, we were able to derive new human induced pluripotent stem cell (hiPSC lines on JAR matrix and show that adhesion of the early hiPSC colonies to JAR matrix is more efficient than to matrigel. In summary, JAR matrix provides a cost-effective and easy-to-prepare alternative for human pluripotent stem cell culture and differentiation. In addition, this matrix is ideal for the efficient generation of new hiPSC lines.

  13. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Directory of Open Access Journals (Sweden)

    Tali Mass

    Full Text Available The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag~4, the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  14. Isolated Cells of Porphyra yezoensis Cultured on Solid Medium

    Institute of Scientific and Technical Information of China (English)

    沈颂东; 戴继勋


    Vegetative cells of Porphyra yezoensis are isolated with sea snail enzyme and cultured on the solidified agar medium. The results of experiments show that the isolated cells can survive,divide and regenerate well on the medium solidified with agar. The first division on the solid medium starts after 7 days' culture, 4 days later than the liquid culture. The survival rate of isolated cells is 71.3% on the solid medium, lower than the 86.2% of that in seawater.Thalli, thalloids,conchocelis, spermatangia and multicellular masses are developed on the solid/medium in the first month, slowly but normally. Spermatangia sacs disappear within 4 weeks. Without adding nutrient liquid onto the surface of solid medium or injecting seawater under the agar layer in order to keep moisture, the thalli and cell groups release monospores to form new thalli instead of enlarging their areas after 5 weeks' culturing. Some monospores regenerate new thalli. Other monospores lose their pigments and minimize their volume and divide quickly to form light pink calli. After 16 weeks, numerous calli can be seen on the solid medium and after 24 weeks' culturing, almost only calli and conchocelis can be seen. If the calli are immersed in seawater, the monospores are released and may develop into young thallus.

  15. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yohei Hayashi


    Full Text Available In recent years, as human pluripotent stem cells (hPSCs have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK, which transmits ECM-integrin signaling to AKT (also known as protein kinase B, has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.

  16. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha


    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  17. Lingual Epithelial Stem Cells and Organoid Culture of Them. (United States)

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo


    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  18. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul


    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

  19. Zinc uptake by brain cells: `surface' versus `bulk' (United States)

    DeStasio, Gelsomina; Pochon, S.; Lorusso, G. F.; Tonner, B. P.; Mercanti, Delio; Ciotti, M. Teresa; Oddo, Nino; Galli, Paolo; Perfetti, P.; Margaritondo, G.


    The uptake of zinc by cerebellar rat cultures upon exposure to 0022-3727/29/8/023/img12 solutions was comparatively investigated using two well known condensed matter physics techniques: synchrotron photoelectron spectromicroscopy and inductively coupled plasma atomic emission spectroscopy. The objective was to apply a strategy - well known in surface physics - to distinguish between `surface' and `bulk' phenomena. The results clearly demonstrate that exposure significantly enhances the bulk (cell cytoplasm) Zn concentration with respect to the physiological level, whereas the effect on the surface (cell membrane) is negligible.

  20. Feline Neural Progenitor Cells I: Long-Term Expansion under Defined Culture Conditions

    Directory of Open Access Journals (Sweden)

    Jing Yang


    Full Text Available Neural progenitor cells (NPCs of feline origin (cNPCs have demonstrated utility in transplantation experiments, yet are difficult to grow in culture beyond the 1 month time frame. Here we use an enriched, serum-free base medium (Ultraculture and report the successful long-term propagation of these cells. Primary cultures were derived from fetal brain tissue and passaged in DMEM/F12-based or Ultraculture-based proliferation media, both in the presence of EGF + bFGF. Cells in standard DMEM/F12-based medium ceased to proliferate by 1-month, whereas the cells in the Ultraculture-based medium continued to grow for at least 5 months (end of study with no evidence of senescence. The Ultraculture-based cultures expressed lower levels of progenitor and lineage-associated markers under proliferation conditions but retained multipotency as evidenced by the ability to differentiate into neurons and glia following growth factor removal in the presence of FBS. Importantly, later passage cNPCs did not develop chromosomal aberrations.

  1. Imipramine induces brain-derived neurotrophic factor mRNA expression in cultured astrocytes. (United States)

    Takano, Katsura; Yamasaki, Hiroshi; Kawabe, Kenji; Moriyama, Mitsuaki; Nakamura, Yoichi


    Depression is one of the most prevalent and livelihood-threatening forms of mental illnesses and the neural circuitry underlying depression remains incompletely understood. Recent studies suggest that the neuronal plasticity involved with brain-derived neurotrophic factor (BDNF) plays an important role in the recovery from depression. Some antidepressants are reported to induce BDNF expression in vivo; however, the mechanisms have been considered solely in neurons and not fully elucidated. In the present study, we evaluated the effects of imipramine, a classic tricyclic antidepressant drug, on BDNF expression in cultured rat brain astrocytes. Imipramine dose-dependently increased BDNF mRNA expression in astrocytes. The imipramine-induced BDNF increase was suppressed with inhibitors for protein kinase A (PKA) or MEK/ERK. Moreover, imipramine exposure activated transcription factor cAMP response element binding protein (CREB) in a dose-dependent manner. These results suggested that imipramine induced BDNF expression through CREB activation via PKA and/or ERK pathways. Imipramine treatment in depression might exert antidepressant action through BDNF production from astrocytes, and glial BDNF expression might be a target of developing novel antidepressants.

  2. Effects of rotational culture on morphology, nitric oxide production and cell cycle of endothelial cells. (United States)

    Tang, Chaojun; Wu, Xue; Ye, Linqi; Xie, Xiang; Wang, Guixue


    Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.

  3. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin;


    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture...

  4. Modulation of Microglial Cell Fcγ Receptor Expression Following Viral Brain Infection (United States)

    Chauhan, Priyanka; Hu, Shuxian; Sheng, Wen S.; Prasad, Sujata; Lokensgard, James R.


    Fcγ receptors (FcγRs) for IgG couple innate and adaptive immunity through activation of effector cells by antigen-antibody complexes. We investigated relative levels of activating and inhibitory FcγRs on brain-resident microglia following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that activating FcγRs were expressed maximally at 5 d post-infection (dpi), while the inhibitory receptor (FcγRIIB) remained highly elevated during both acute and chronic phases of infection. The highly induced expression of activating FcγRIV during the acute phase of infection was also noteworthy. Furthermore, in vitro analysis using cultured primary microglia demonstrated the role of interferon (IFN)γ and interleukin (IL)-4 in polarizing these cells towards a M1 or M2 phenotype, respectively. Microglial cell-polarization correlated with maximal expression of either FcγRIV or FcγRIIB following stimulation with IFNγ or IL-4, respectively. Finally, we observed a significant delay in polarization of microglia towards an M2 phenotype in the absence of FcγRs in MCMV-infected Fcer1g and FcgR2b knockout mice. These studies demonstrate that neuro-inflammation following viral infection increases expression of activating FcγRs on M1-polarized microglia. In contrast, expression of the inhibitory FcγRIIB receptor promotes M2-polarization in order to shut-down deleterious immune responses and limit bystander brain damage. PMID:28165503

  5. A glucose fuel cell for implantable brain-machine interfaces.

    Directory of Open Access Journals (Sweden)

    Benjamin I Rapoport

    Full Text Available We have developed an implantable fuel cell that generates power through glucose oxidation, producing 3.4 μW cm(-2 steady-state power and up to 180 μW cm(-2 peak power. The fuel cell is manufactured using a novel approach, employing semiconductor fabrication techniques, and is therefore well suited for manufacture together with integrated circuits on a single silicon wafer. Thus, it can help enable implantable microelectronic systems with long-lifetime power sources that harvest energy from their surrounds. The fuel reactions are mediated by robust, solid state catalysts. Glucose is oxidized at the nanostructured surface of an activated platinum anode. Oxygen is reduced to water at the surface of a self-assembled network of single-walled carbon nanotubes, embedded in a Nafion film that forms the cathode and is exposed to the biological environment. The catalytic electrodes are separated by a Nafion membrane. The availability of fuel cell reactants, oxygen and glucose, only as a mixture in the physiologic environment, has traditionally posed a design challenge: Net current production requires oxidation and reduction to occur separately and selectively at the anode and cathode, respectively, to prevent electrochemical short circuits. Our fuel cell is configured in a half-open geometry that shields the anode while exposing the cathode, resulting in an oxygen gradient that strongly favors oxygen reduction at the cathode. Glucose reaches the shielded anode by diffusing through the nanotube mesh, which does not catalyze glucose oxidation, and the Nafion layers, which are permeable to small neutral and cationic species. We demonstrate computationally that the natural recirculation of cerebrospinal fluid around the human brain theoretically permits glucose energy harvesting at a rate on the order of at least 1 mW with no adverse physiologic effects. Low-power brain-machine interfaces can thus potentially benefit from having their implanted units

  6. Immune cell trafficking from the brain maintains CNS immune tolerance. (United States)

    Mohammad, Mohammad G; Tsai, Vicky W W; Ruitenberg, Marc J; Hassanpour, Masoud; Li, Hui; Hart, Prue H; Breit, Samuel N; Sawchenko, Paul E; Brown, David A


    In the CNS, no pathway dedicated to immune surveillance has been characterized for preventing the anti-CNS immune responses that develop in autoimmune neuroinflammatory disease. Here, we identified a pathway for immune cells to traffic from the brain that is associated with the rostral migratory stream (RMS), which is a forebrain source of newly generated neurons. Evaluation of fluorescently labeled leukocyte migration in mice revealed that DCs travel via the RMS from the CNS to the cervical LNs (CxLNs), where they present antigen to T cells. Pharmacologic interruption of immune cell traffic with the mononuclear cell-sequestering drug fingolimod influenced anti-CNS T cell responses in the CxLNs and modulated experimental autoimmune encephalomyelitis (EAE) severity in a mouse model of multiple sclerosis (MS). Fingolimod treatment also induced EAE in a disease-resistant transgenic mouse strain by altering DC-mediated Treg functions in CxLNs and disrupting CNS immune tolerance. These data describe an immune cell pathway that originates in the CNS and is capable of dampening anti-CNS immune responses in the periphery. Furthermore, these data provide insight into how fingolimod treatment might exacerbate CNS neuroinflammation in some cases and suggest that focal therapeutic interventions, outside the CNS have the potential to selectively modify anti-CNS immunity.

  7. Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer. (United States)

    Pathak, Sen; Multani, Asha S; Banerji, Pratip; Banerji, Prasanta


    Although conventional chemotherapies are used to treat patients with malignancies, damage to normal cells is problematic. Blood-forming bone marrow cells are the most adversely affected. It is therefore necessary to find alternative agents that can kill cancer cells but have minimal effects on normal cells. We investigated the brain cancer cell-killing activity of a homeopathic medicine, Ruta, isolated from a plant, Ruta graveolens. We treated human brain cancer and HL-60 leukemia cells, normal B-lymphoid cells, and murine melanoma cells in vitro with different concentrations of Ruta in combination with Ca3(PO4)2. Fifteen patients diagnosed with intracranial tumors were treated with Ruta 6 and Ca3(PO4)2. Of these 15 patients, 6 of the 7 glioma patients showed complete regression of tumors. Normal human blood lymphocytes, B-lymphoid cells, and brain cancer cells treated with Ruta in vitro were examined for telomere dynamics, mitotic catastrophe, and apoptosis to understand the possible mechanism of cell-killing, using conventional and molecular cytogenetic techniques. Both in vivo and in vitro results showed induction of survival-signaling pathways in normal lymphocytes and induction of death-signaling pathways in brain cancer cells. Cancer cell death was initiated by telomere erosion and completed through mitotic catastrophe events. We propose that Ruta in combination with Ca3(PO4)2 could be used for effective treatment of brain cancers, particularly glioma.

  8. Cell sources for in vitro human liver cell culture models. (United States)

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny


    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

  9. Derivation of injury-responsive dendritic cells for acute brain targeting and therapeutic protein delivery in the stroke-injured rat.

    Directory of Open Access Journals (Sweden)

    Nathan C Manley

    Full Text Available Research with experimental stroke models has identified a wide range of therapeutic proteins that can prevent the brain damage caused by this form of acute neurological injury. Despite this, we do not yet have safe and effective ways to deliver therapeutic proteins to the injured brain, and this remains a major obstacle for clinical translation. Current targeted strategies typically involve invasive neurosurgery, whereas systemic approaches produce the undesirable outcome of non-specific protein delivery to the entire brain, rather than solely to the injury site. As a potential way to address this, we developed a protein delivery system modeled after the endogenous immune cell response to brain injury. Using ex-vivo-engineered dendritic cells (DCs, we find that these cells can transiently home to brain injury in a rat model of stroke with both temporal and spatial selectivity. We present a standardized method to derive injury-responsive DCs from bone marrow and show that injury targeting is dependent on culture conditions that maintain an immature DC phenotype. Further, we find evidence that when loaded with therapeutic cargo, cultured DCs can suppress initial neuron death caused by an ischemic injury. These results demonstrate a non-invasive method to target ischemic brain injury and may ultimately provide a way to selectively deliver therapeutic compounds to the injured brain.

  10. Polyamines in relation to growth in carrot cell cultures. (United States)

    Fallon, K M; Phillips, R


    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

  11. Polyamines in Relation to Growth in Carrot Cell Cultures 1 (United States)

    Fallon, Kevin M.; Phillips, Richard


    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID:16666271

  12. A self-feeding roller bottle for continuous cell culture. (United States)

    Berson, R Eric; Friederichs, Goetz


    The concept of a self-feeding roller bottle that delivers a continuous supply of fresh media to cells in culture, which is mechanically simplistic and works with existing roller apparatuses, is presented here. A conventional roller bottle is partitioned into two chambers; one chamber contains the fresh culture media reservoir, and the other contains the cell culture chamber. A spiroid of tubing inside the fresh media reservoir acts as a pump when the bottle rotates on its horizontal axis, continuously delivering fresh media through an opening in the partition to the cell culture chamber. The modified bottle proved capable of maintaining steady-state cell densities of a hybridoma cell line over the 10-day period tested, although at lower densities than reached during batch operation due to the continuous volume dilution. Steady-state density proved to be controllable by adjusting the perfusion rate, which changes with the rotation rate of the bottle. Specific antibody production rate is as much as 3.7 times the rate in conventional roller bottles operating with intermittent batch feeding.

  13. Cell culture systems for the hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Gilles Duverlie; Czeslaw Wychowski


    Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consensus clones have been selected and established in a human hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these replicons did not support production of infectious virus. Interestingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.

  14. A single-cell and feeder-free culture system for monkey embryonic stem cells. (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya


    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  15. Acquired resistance to auranofin in cultured human cells. (United States)

    Glennås, A; Rugstad, H E


    A substrain (HEAF) of cultured human epithelial cells, grown as monolayers, was selected for resistance to auranofin (AF), a gold-containing anti-arthritic drug, by growing the parental HE cells with stepwise increased concentrations of AF in the medium. HEAF cells acquired resistance to 2 mumol AF/l, twice the concentration tolerated by the sensitive HE cells. Resistance to AF was also demonstrated in another substrain (HE100) originally selected for by its cadmium resistance, and characterized by a high cytosolic metallothionein (MT) content. Following continuous exposure to 2 mumol AF/l for 4 days, 58% of the HEAF cells, 67% of the HE100 cells, and 16% of the HE cells remained adherent to the flasks, compared with non-treated controls. Following 24 h AF exposure to living cells, HEAF cells had one-half and HE100 cells twice the cellular and cytosolic gold concentration per mg protein, as compared with HE cells. Gel filtration of cell cytosols revealed gold-binding proteins with a mol. wt. of about 10 000 apparently occurring on AF exposure in HEAF and HE cells. They bound 10-15% of cytosolic gold. MT in HE100 cells bound AF-gold to about the same extent. We suggest that the ability of cells to maintain the gold concentration at a low level (HEAF) and trapping of gold by MT (HE100) or low molecular weight proteins occurring on AF treatment (HEAF) may be mechanisms contributing to the observed cellular resistance to AF.

  16. Furan fatty acids efficiently rescue brain cells from cell death induced by oxidative stress. (United States)

    Teixeira, Antoinette; Cox, Ruud C; Egmond, Maarten R


    Treatment of rat brain C6 astroglioma cells with furan fatty acid F6 prior to exposure to hydrogen peroxide shows a strong protective effect of F6 against cell death resulting from oxidative stress. This protective effect is obtained only for F6 administered as a free fatty acid and with an intact furan ring. It is proposed that brain cells are rescued by F6 scavenging radicals elicited by lipid peroxidation within the cell membrane. Oxidative processes outside the cell membrane, such as protein carbonylation, are not affected by F6. Furan fatty acids such as those present in fish oils and marine organisms are likely beneficial for consumption in reducing the risk of diseases that have been implicated to arise from oxidative stress, such as Alzheimer's disease.

  17. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

    Directory of Open Access Journals (Sweden)

    Tzuu-Yuan Huang


    Full Text Available Demethoxycurcumin (DMC; a curcumin-related demethoxy compound has been recently shown to display antioxidant and antitumor activities. It has also produced a potent chemopreventive action against cancer. In the present study, the antiproliferation (using the MTT assay, DMC was found to have cytotoxic activities against GBM 8401 cell with IC50 values at 22.71 μM and induced apoptosis effects of DMC have been investigated in human brain malignant glioma GBM 8401 cells. We have studied the mitochondrial membrane potential (MMP, DNA fragmentation, caspase activation, and NF-κB transcriptional factor activity. By these approaches, our results indicated that DMC has produced an inhibition of cell proliferation as well as the activation of apoptosis in GBM 8401 cells. Both effects were observed to increase in proportion with the dosage of DMC treatment, and the apoptosis was induced by DMC in human brain malignant glioma GBM 8401 cells via mitochondria- and caspase-dependent pathways.

  18. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin


    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  19. Cell culture media impact on drug product solution stability. (United States)

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J


    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016.

  20. Language/Culture Modulates Brain and Gaze Processes in Audiovisual Speech Perception (United States)

    Hisanaga, Satoko; Sekiyama, Kaoru; Igasaki, Tomohiko; Murayama, Nobuki


    Several behavioural studies have shown that the interplay between voice and face information in audiovisual speech perception is not universal. Native English speakers (ESs) are influenced by visual mouth movement to a greater degree than native Japanese speakers (JSs) when listening to speech. However, the biological basis of these group differences is unknown. Here, we demonstrate the time-varying processes of group differences in terms of event-related brain potentials (ERP) and eye gaze for audiovisual and audio-only speech perception. On a behavioural level, while congruent mouth movement shortened the ESs’ response time for speech perception, the opposite effect was observed in JSs. Eye-tracking data revealed a gaze bias to the mouth for the ESs but not the JSs, especially before the audio onset. Additionally, the ERP P2 amplitude indicated that ESs processed multisensory speech more efficiently than auditory-only speech; however, the JSs exhibited the opposite pattern. Taken together, the ESs’ early visual attention to the mouth was likely to promote phonetic anticipation, which was not the case for the JSs. These results clearly indicate the impact of language and/or culture on multisensory speech processing, suggesting that linguistic/cultural experiences lead to the development of unique neural systems for audiovisual speech perception. PMID:27734953

  1. Effects of Visible Light on Cultured Bovine Trabecular Cells

    Institute of Scientific and Technical Information of China (English)

    姜发纲; 郝风芹; 魏厚仁; 许德胜


    To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1. 12 mW/cm2 , the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

  2. Stem cell-paved biobridges facilitate stem transplant and host brain cell interactions for stroke therapy. (United States)

    Duncan, Kelsey; Gonzales-Portillo, Gabriel S; Acosta, Sandra A; Kaneko, Yuji; Borlongan, Cesar V; Tajiri, Naoki


    Distinguished by an infarct core encased within a penumbra, stroke remains a primary source of mortality within the United States. While our scientific knowledge regarding the pathology of stroke continues to improve, clinical treatment options for patients suffering from stroke are extremely limited. Tissue plasminogen activator (tPA) remains the sole FDA-approved drug proven to be helpful following stroke. However, due to the need to administer the drug within 4.5h of stroke onset its usefulness is constrained to less than 5% of all patients suffering from ischemic stroke. One experimental therapy for the treatment of stroke involves the utilization of stem cells. Stem cell transplantation has been linked to therapeutic benefit by means of cell replacement and release of growth factors; however the precise means by which this is accomplished has not yet been clearly delineated. Using a traumatic brain injury model, we recently demonstrated the ability of transplanted mesenchymal stromal cells (MSCs) to form a biobridge connecting the area of injury to the neurogenic niche within the brain. We hypothesize that MSCs may also have the capacity to create a similar biobridge following stroke; thereby forming a conduit between the neurogenic niche and the stroke core and peri-infarct area. We propose that this biobridge could assist and promote interaction of host brain cells with transplanted stem cells and offer more opportunities to enhance the effectiveness of stem cell therapy in stroke. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke.

  3. Xeno-free culture of human pluripotent stem cells. (United States)

    Bergström, Rosita; Ström, Susanne; Holm, Frida; Feki, Anis; Hovatta, Outi


    Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined with their potential to treat human diseases have accelerated the drive to develop xenogenic-free, chemically defined culture systems that support pluripotent self-renewal and directed differentiation. In this chapter, we describe four xeno-free culture systems that have been successful in supporting undifferentiated growth of hPSCs as well as methods for xeno-free subculture and cryopreservation of hPSCs. Each culture system consists of a xeno-free growth medium and xeno-free substratum: (1) TeSR2™ with human recombinant laminin (LN-511); (2) NutriStem™ with LN-511; (3) RegES™ with human foreskin fibroblasts (hFFs); (4) KO-SR Xeno-Free™/GF cocktail with CELLstart™ matrix.

  4. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies. (United States)

    Danto, S I; Zabski, S M; Crandall, E D


    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  5. Measurement and analysis of calcium signaling in heterogeneous cell cultures. (United States)

    Richards, Gillian R; Jack, Andrew D; Platts, Amy; Simpson, Peter B


    High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

  6. Chikungunya virus isolation using simplified cell culture technique in Mauritius. (United States)

    Pyndiah, M N; Pursem, V; Meetoo, G; Daby, S; Ramuth, V; Bhinkah, P; Chuttoo, R; Paratian, U


    During the chikungunya outbreak of 2005 - 2006, the only laboratory facilities available in Mauritius were virus isolation in cell culture tubes and serology. The laboratory was submerged with large numbers of blood samples. Comparative isolation was made in human embryonic lung (HEL) and VERO cells grown in 96-well plate. Culture on HEL cells was found to be more sensitive and presence of cytopathic effect (CPE) was observed earlier than in VERO cells. Out of the 18 300 blood samples inoculated on HEL, 11 165 were positive. This virus isolation method was of great help for the surveillance and control of the vectors. In cases of an outbreak a cheap, rapid and simple method of isolating chikungunya virus is described.

  7. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;


    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  8. Cell cavities increase tortuosity in brain extracellular space. (United States)

    Tao, A; Tao, L; Nicholson, C


    Brain extracellular space (ECS) forms hindered pathways for molecular diffusion in chemical signaling and drug delivery. Hindrance is quantified by the tortuosity lambda; the tortuosity obtained from simulations using uniformly spaced convex cells is significantly lower than that measured experimentally. To attempt to account for the difference in results, this study employed a variety of ECS models based on an array of cubic cells containing open rectangular cavities that provided the ECS with dead-space microdomains. Monte Carlo simulations demonstrated that, in such ECS models, lambda can equal or exceed the typical experimental value of about 1.6. The simulations further revealed that lambda is relatively independent of cavity shape and the number of cavities per cell. It mainly depends on the total ECS volume fraction alpha, the cavity volume fraction alpha(c), and whether the cavity is located at the center of a cell face or formed at the junction of multiple cells. To describe the results from the different ECS models, an expression was obtained that related lambda to alpha, alpha(c), and an empirical exit factor beta that correlated with the ease with which a molecule could leave a cavity and its vicinity.

  9. Nootropic agents stimulate neurogenesis. Brain Cells, Inc.: WO2007104035. (United States)

    Taupin, Philippe


    The application is in the field of adult neurogenesis, neural stem cells and cellular therapy. It aims to characterize the activity of nootropic agents on adult neurogenesis in vitro. Nootropic agents are substances improving cognitive and mental abilities. AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) and nootropic agents were assessed for the potential to differentiate human neural progenitor and stem cells into neuronal cells in vitro. They were also tested for their behavioural activity on the novel object recognition task. AMPA, piracetam, FK-960 and SGS-111 induce and stimulate neuronal differentiation of human-derived neural progenitor and stem cells. SGS-111 increases the number of visits to the novel object. The neurogenic activity of piracetam and SGS-111 is mediated through AMPA receptor. The neurogenic activity of SGS-111 may contribute and play a role in its nootropic activity. These results suggest that nootropic agents may elicit some of their effects through their neurogenic activity. The application claims the use of nootropic agents for their neurogenic activity and for the treatment of neurological diseases, disorders and injuries, by stimulating or increasing the generation of neuronal cells in the adult brain.

  10. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency. (United States)

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer


    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  11. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    Directory of Open Access Journals (Sweden)

    Tor Paaske Utheim


    Full Text Available The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC, which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD. Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  12. Brain-derived neurotrophic factor mediates the activity-dependent regulation of inhibition in neocortical cultures. (United States)

    Rutherford, L C; DeWan, A; Lauer, H M; Turrigiano, G G


    The excitability of cortical circuits is modulated by interneurons that release the inhibitory neurotransmitter GABA. In primate and rodent visual cortex, activity deprivation leads to a decrease in the expression of GABA. This suggests that activity is able to adjust the strength of cortical inhibition, but this has not been demonstrated directly. In addition, the nature of the signal linking activity to GABA expression has not been determined. Activity is known to regulate the expression of the neurotrophin brain-derived neurotrophic factor (BDNF), and BDNF has been shown to influence the phenotype of GABAergic interneurons. We use a culture system from postnatal rat visual cortex to test the hypothesis that activity is regulating the strength of cortical inhibition through the regulation of BDNF. Cultures were double-labeled against GABA and the neuronal marker MAP2, and the percentage of neurons that were GABA-positive was determined. Blocking spontaneous activity in these cultures reversibly decreased the number of GABA-positive neurons without affecting neuronal survival. Voltage-clamp analysis of inhibitory currents demonstrated that activity blockade also decreased GABA-mediated inhibition onto pyramidal neurons and raised pyramidal neuron firing rates. All of these effects were prevented by incubation with BDNF during activity blockade, but not by neurotrophin 3 or nerve growth factor. Additionally, blockade of neurotrophin signaling mimicked the effects of activity blockade on GABA expression. These data suggest that activity regulates cortical inhibition through a BDNF-dependent mechanism and that this neurotrophin plays an important role in the control of cortical excitability.

  13. Withaferin A from cell cultures of Withania somnifera

    Directory of Open Access Journals (Sweden)

    Ciddi Veeresham


    Full Text Available Suspension cultures of Withania somnifera cells were established and shown to produce withaferin A. The identification of withaferin A was done by TLC, UV absorption, HPLC and electron spray mass spectroscopy. These cultures could be strongly elicited by exposure to salacin. Addition of salacin at the concentration of 750 µM to the cultures in production medium enhanced production levels of withaferin A to 25±2.9 mg/l compared to 0.47±0.03 mg/l in unelicited controls. This report is the first to demonstrate withaferin A production in plant suspension cultures and provides prerequisites for commercial scale, controlled production of withaferin A.

  14. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.


    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  15. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources (United States)

    Lippmann, Ethan S.; Al-Ahmad, Abraham; Azarin, Samira M.; Palecek, Sean P.; Shusta, Eric V.


    Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

  16. Crude subcellular fractionation of cultured mammalian cell lines


    Holden Paul; Horton William A


    Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this...

  17. Intracranial Tumor Cell Migration and the Development of Multiple Brain Metastases in Malignant Melanoma

    Directory of Open Access Journals (Sweden)

    Trude G. Simonsen


    Full Text Available INTRODUCTION: A majority of patients with melanoma brain metastases develop multiple lesions, and these patients show particularly poor prognosis. To develop improved treatment strategies, detailed insights into the biology of melanoma brain metastases, and particularly the development of multiple lesions, are needed. The purpose of this preclinical investigation was to study melanoma cell migration within the brain after cell injection into a well-defined intracerebral site. METHODS: A-07, D-12, R-18, and U-25 human melanoma cells transfected with green fluorescent protein were injected stereotactically into the right cerebral hemisphere of nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or histological examination. RESULTS: Intracerebral inoculation of melanoma cells produced multiple lesions involving all regions of the brain, suggesting that the cells were able to migrate over substantial distances within the brain. Multiple modes of transport were identified, and all transport modes were observed in all four melanoma lines. Thus, the melanoma cells were passively transported via the flow of cerebrospinal fluid in the meninges and ventricles, they migrated actively along leptomeningeal and brain parenchymal blood vessels, and they migrated actively along the surfaces separating different brain compartments. CONCLUSION: Migration of melanoma cells after initial arrest, extravasation, and growth at a single location within the brain may contribute significantly to the development of multiple melanoma brain metastases.

  18. Breaking the Blood-Brain Barrier With Mannitol to Aid Stem Cell Therapeutics in the Chronic Stroke Brain. (United States)

    Tajiri, Naoki; Lee, Jea Young; Acosta, Sandra; Sanberg, Paul R; Borlongan, Cesar V


    Blood-brain barrier (BBB) permeabilizers, such as mannitol, can facilitate peripherally delivered stem cells to exert therapeutic benefits on the stroke brain. Although this BBB permeation-aided stem cell therapy has been demonstrated in the acute stage of stroke, such BBB permeation in the chronic stage of the disease remains to be examined. Adult Sprague-Dawley rats initially received sham surgery or experimental stroke via the 1-h middle cerebral artery occlusion (MCAo) model. At 1 month after the MCAo surgery, stroke animals were randomly assigned to receive human umbilical cord stem cells only (2 million viable cells), mannitol only (1.1 mol/L mannitol at 4°C), combined human umbilical cord stem cells (200,000 viable cells) and mannitol (1.1 mol/L mannitol at 4°C), and vehicle (phosphate-buffered saline) only. Stroke animals that received human umbilical cord blood cells alone or combined human umbilical cord stem cells and mannitol exhibited significantly improved motor performance and significantly better brain cell survival in the peri-infarct area compared to stroke animals that received vehicle or mannitol alone, with mannitol treatment reducing the stem cell dose necessary to afford functional outcomes. Enhanced neurogenesis in the subventricular zone accompanied the combined treatment of human umbilical cord stem cells and mannitol. We showed that BBB permeation facilitates the therapeutic effects of a low dose of peripherally transplanted stem cells to effectively cause functional improvement and increase neurogenesis in chronic stroke.

  19. Brain-derived Neurotrophic Factor Promotes the Migration of Olfactory Ensheathing Cells Through TRPC Channels. (United States)

    Wang, Ying; Teng, Hong-Lin; Gao, Yuan; Zhang, Fan; Ding, Yu-Qiang; Huang, Zhi-Hui


    Olfactory ensheathing cells (OECs) are a unique type of glial cells with axonal growth-promoting properties in the olfactory system. Organized migration of OECs is essential for neural regeneration and olfactory development. However, the molecular mechanism of OEC migration remains unclear. In the present study, we examined the effects of brain-derived neurotrophic factor (BDNF) on OEC migration. Initially, the "scratch" migration assay, the inverted coverslip and Boyden chamber migration assays showed that BDNF could promote the migration of primary cultured OECs. Furthermore, BDNF gradient attracted the migration of OECs in single-cell migration assays. Mechanistically, TrkB receptor expressed in OECs mediated BDNF-induced OEC migration, and BDNF triggered calcium signals in OECs. Finally, transient receptor potential cation channels (TRPCs) highly expressed in OECs were responsible for BDNF-induced calcium signals, and required for BDNF-induced OEC migration. Taken together, these results demonstrate that BDNF promotes the migration of cultured OECs and an unexpected finding is that TRPCs are required for BDNF-induced OEC migration. GLIA 2016;64:2154-2165.

  20. A biofidelic 3D culture model to study the development of brain cellular systems (United States)

    Ren, M.; Du, C.; Herrero Acero, E.; Tang-Schomer, M. D.; Özkucur, N.


    Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (Vmem) according to methods described in a previous work that implicated Ivm in the arrangement and connectivity of cortical cell assemblies. The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the dev