Sample records for characterizing tissue culture

  1. Characterization of human myoblast cultures for tissue engineering. (United States)

    Stern-Straeter, Jens; Bran, Gregor; Riedel, Frank; Sauter, Alexander; Hörmann, Karl; Goessler, Ulrich Reinhart


    Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation

  2. Whole genome characterization of non-tissue culture adapted HRSV strains in severely infected children

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    Kumaria Rajni


    Full Text Available Abstract Background Human respiratory syncytial virus (HRSV is the most important virus causing lower respiratory infection in young children. The complete genetic characterization of RSV clinical strains is a prerequisite for understanding HRSV infection in the clinical context. Current information about the genetic structure of the HRSV genome has largely been obtained using tissue culture adapted viruses. During tissue culture adaptation genetic changes can be introduced into the virus genome, which may obscure subtle variations in the genetic structure of different RSV strains. Methods In this study we describe a novel Sanger sequencing strategy which allowed the complete genetic characterisation of 14 clinical HRSV strains. The viruses were sequenced directly in the nasal washes of severely hospitalized children, and without prior passage of the viruses in tissue culture. Results The analysis of nucleotide sequences suggested that vRNA length is a variable factor among primary strains, while the phylogenetic analysis suggests selective pressure for change. The G gene showed the greatest sequence variation (2-6.4%, while small hydrophobic protein and matrix genes were completely conserved across all clinical strains studied. A number of sequence changes in the F, L, M2-1 and M2-2 genes were observed that have not been described in laboratory isolates. The gene junction regions showed more sequence variability, and in particular the intergenic regions showed a highest level of sequence variation. Although the clinical strains grew slower than the HRSVA2 virus isolate in tissue culture, the HRSVA2 isolate and clinical strains formed similar virus structures such as virus filaments and inclusion bodies in infected cells; supporting the clinical relevance of these virus structures. Conclusion This is the first report to describe the complete genetic characterization of HRSV clinical strains that have been sequenced directly from clinical

  3. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

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    Mehta Bhavya C


    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  4. Plant tissue culture techniques


    Rolf Dieter Illg


    Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.

  5. Plant tissue culture techniques

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    Rolf Dieter Illg


    Full Text Available Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus or organized tissues or organs put in culture, under controlled sterile conditions.

  6. Mass spectrometric characterization of elements and molecules in cell cultures and tissues

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    Arlinghaus, H.F. [Physikalisches Institut, Universitaet Muenster, Wilhelm-Klemm-Str. 10, D-48149 Muenster (Germany)]. E-mail:; Kriegeskotte, C. [Physikalisches Institut, Universitaet Muenster, Wilhelm-Klemm-Str. 10, D-48149 Muenster (Germany); Fartmann, M. [Physikalisches Institut, Universitaet Muenster, Wilhelm-Klemm-Str. 10, D-48149 Muenster (Germany); Wittig, A. [Strahlenklinik, Universitaetsklinikum Essen, D-45122 Essen (Germany); Sauerwein, W. [Strahlenklinik, Universitaetsklinikum Essen, D-45122 Essen (Germany); Lipinsky, D. [Physikalisches Institut, Universitaet Muenster, Wilhelm-Klemm-Str. 10, D-48149 Muenster (Germany)


    Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (laser-SNMS) have been used to image and quantify targeted compounds, intrinsic elements and molecules with subcellular resolution in single cells of both cell cultures and tissues. Special preparation procedures for analyzing cell cultures and tissue materials were developed. Cancer cells type MeWo, incubated with boronated compounds, were sandwiched between two substrates, cryofixed, freeze-fractured and freeze-dried. Also, after injection with boronated compounds, different types of mouse tissues were extracted, prepared on a special specimen carrier and plunged with high velocity into LN{sub 2}-cooled propane for cryofixation. After trimming, these tissue blocks were freeze-dried. The measurements of the K/Na ratio demonstrated that for both cell cultures and tissue materials the special preparation techniques used were appropriate for preserving the chemical and structural integrity of the living cell. The boron images show inter- and intracellular boron signals with different intensities. Molecular images show distinct features partly correlated with the cell structure. A comparison between laser-SNMS and ToF-SIMS showed that especially laser-SNMS is particularly well-suited for identifying specific cell structures and imaging ultratrace element concentrations in tissues.

  7. Isolation, Culturing, Characterization and Aging of Adipose Tissue-derived Mesenchymal Stem Cells: A Brief Overview

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    Ezzatollah Fathi


    Full Text Available ABSTRACT The aim of this review was to describe the current state-of-the-art regarding isolation, characterization and aging of adipose tissue-derived mesenchymal stem cells (ADSCs. Mesenchymal stem cells (MSCs have recently received widespread attention because of their potential use in tissue-engineering applications. Various studies have indicated that MSCs with a fibroblast-like morphology migrate to the sites of injury and help to regenerate damaged tissue. Over the past few years, it has been recognized that fat is not only an energy supply, but also a rich source of multipotent stem cells that can be easily harvested, isolated and selected as compared with other tissues. ADSCs are particularly interesting because of their rapid proliferation and multidirectional differentiation potential.

  8. Norway spruce (Picea abies) laccases: characterization of a laccase in a lignin-forming tissue culture. (United States)

    Koutaniemi, Sanna; Malmberg, Heli A; Simola, Liisa K; Teeri, Teemu H; Kärkönen, Anna


    Secondarily thickened cell walls of water-conducting vessels and tracheids and support-giving sclerenchyma cells contain lignin that makes the cell walls water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five full-length laccase cDNAs from developing xylem and an extracellular lignin-forming cell culture of spruce. In addition, we purified and biochemically characterized one culture medium laccase from the lignin-forming cell culture. This laccase has an acidic pH optimum (pH 3.8-4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 μM; however, the laccase has a lower catalytic efficiency (V(max)/K(m)) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

  9. Plant Tissue Culture Studies. (United States)

    Smith, Robert Alan

    Plant tissue culture has developed into a valid botanical discipline and is considered a key area of biotechnology, but it has not been a key component of the science curriculum because of the expensive and technical nature of research in this area. This manual presents a number of activities that are relatively easy to prepare and perform. The…

  10. Characterization of the Embryogenic Tissue of the Norway Spruce Including a Transition Layer between the Tissue and the Culture Medium by Magnetic Resonance Imaging (United States)

    Kořínek, R.; Mikulka, J.; Hřib, J.; Hudec, J.; Havel, L.; Bartušek, K.


    The paper describes the visualization of the cells (ESEs) and mucilage (ECMSN) in an embryogenic tissue via magnetic resonance imaging (MRI) relaxometry measurement combined with the subsequent multi-parametric segmentation. The computed relaxometry maps T1 and T2 show a thin layer (transition layer) between the culture medium and the embryogenic tissue. The ESEs, mucilage, and transition layer differ in their relaxation times T1 and T2; thus, these times can be used to characterize the individual parts within the embryogenic tissue. The observed mean values of the relaxation times T1 and T2 of the ESEs, mucilage, and transition layer are as follows: 1469 ± 324 and 53 ± 10 ms, 1784 ± 124 and 74 ± 8 ms, 929 ± 164 and 32 ± 4.7 ms, respectively. The multi-parametric segmentation exploiting the T1 and T2 relaxation times as a classifier shows the distribution of the ESEs and mucilage within the embryogenic tissue. The discussed T1 and T2 indicators can be utilized to characterize both the growth-related changes in an embryogenic tissue and the effect of biotic/abiotic stresses, thus potentially becoming a distinctive indicator of the state of any examined embryogenic tissue.

  11. Characterization of Human Vaginal Mucosa Cells for Autologous In Vitro Cultured Vaginal Tissue Transplantation in Patients with MRKH Syndrome

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    Cristina Nodale


    Full Text Available Mayer-Rokitansky-Küster-Hauser (MRKH is a rare syndrome characterized by congenital aplasia of the uterus and vagina. The most common procedure used for surgical reconstruction of the neovagina is the McIndoe vaginoplasty, which consists in creation of a vaginal canal covered with a full-thickness skin graft. Here we characterized the autologous in vitro cultured vaginal tissue proposed as alternative material in our developed modified McIndoe vaginoplasty in order to underlie its importance in autologous total vaginal replacement. To this aim human vaginal mucosa cells (HVMs were isolated from vaginal mucosa of patients affected by MRKH syndrome and characterized with respect to growth kinetics, morphology, PAS staining, and expression of specific epithelial markers by immunofluorescence, Western blot, and qRT-PCR analyses. The presence of specific epithelial markers along with the morphology and the presence of mucified cells demonstrated the epithelial nature of HMVs, important for an efficient epithelialization of the neovagina walls and for creating a functional vaginal cavity. Moreover, these cells presented characteristics of effective proliferation as demonstrated by growth kinetics assay. Therefore, the autologous in vitro cultured vaginal tissue might represent a highly promising and valid material for McIndoe vaginoplasty.

  12. NASA Bioreactor tissue culture (United States)


    Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

  13. Tissue culture: the unrealized potential


    Sato, Gordon


    Lack of differentiated functions of the tissue of origin in tissue culture thought to be due to dedifferentiation was shown to be due to selective overgrowth of fibroblasts. Enrichment culture techniques, (alternate animal and culture passage), designed to give the functionally differentiated cells selective advantage over the fibroblasts resulted in a large number of functionally differentiated clonal strains. Thus the dogma of dedifferentiation was destroyed. It is proposed to substitute th...

  14. Biomedical advances from tissue culture


    Okamoto, Tetsuji; Sato, J. Denry; Barnes, David W.; Sato, Gordon H.


    The demonstration that the “dedifferentiation” of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that...

  15. History of plant tissue culture. (United States)

    Thorpe, Trevor


    Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the beginning of the twentieth century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology in the twenty-first century. The historical development of these in vitro technologies and their applications is the focus of this chapter.

  16. Epigenetics in plant tissue culture

    NARCIS (Netherlands)

    Smulders, M.J.M.; Klerk, de G.J.M.


    Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at

  17. Norway spruce (Picea abies) laccases:Characterization of a laccase in a lignin-forming tissue culture

    Institute of Scientific and Technical Information of China (English)

    Sanna Koutaniemi; Heli A Malmberg; Liisa K Simola; Teemu H Teeri; Anna Karkonen


    Secondarily thickened cel wal s of water-conducting vessels and tracheids and support-giving sclerenchyma cel s contain lignin that makes the cel wal s water impermeable and strong. To what extent laccases and peroxidases contribute to lignin biosynthesis in muro is under active evaluation. We performed an in silico study of Norway spruce (Picea abies (L.) Karst.) laccases utilizing available genomic data. As many as 292 laccase encoding sequences (genes, gene fragments, and pseudogenes) were detected in the spruce genome. Out of the 112 genes annotated as laccases, 79 are expressed at some level. We isolated five ful-length laccase cDNAs from developing xylem and an extracel ular lignin-forming cel culture of spruce. In addition, we purified and biochemical y characterized one culture medium laccase from the lignin-forming cel culture. This laccase has an acidic pH optimum (pH 3.8–4.2) for coniferyl alcohol oxidation. It has a high affinity to coniferyl alcohol with an apparent Km value of 3.5 mM;however, the laccase has a lower catalytic efficiency (Vmax/Km) for coniferyl alcohol oxidation compared with some purified culture medium peroxidases. The properties are discussed in the context of the information already known about laccases/coniferyl alcohol oxidases of coniferous plants.

  18. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

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    Funk, C.; Croteau, R. (Washington State Univ., Pullman (United States))


    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  19. Characterizing European cultural landscapes

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    Tieskens, Koen F.; Schulp, Catharina J E; Levers, Christian


    Almost all rural areas in Europe have been shaped or altered by humans and can be considered cultural landscapes, many of which now are considered to entail valuable cultural heritage. Current dynamics in land management have put cultural landscapes under a huge pressure of agricultural intensifi......Almost all rural areas in Europe have been shaped or altered by humans and can be considered cultural landscapes, many of which now are considered to entail valuable cultural heritage. Current dynamics in land management have put cultural landscapes under a huge pressure of agricultural...... intensification and land abandonment. To prevent the loss of cultural landscapes, knowledge on the location of different types of cultural landscapes is needed. In this paper, we present a characterization of European cultural landscapes based on the prevalence of three key dimensions of cultural landscapes......: landscape structure, management intensity, and value and meaning. We mapped these dimensions across Europe at a 1-km resolution by combining proxies on management intensity and landscape structure with new indicators such as social media usage and registered traditional food products. We integrated...

  20. Characterization of a unique OpMNPV-specific early gene not required for viral infection in tissue culture. (United States)

    Shippam, C; Wu, X; Stewart, S; Theilmann, D A


    opep-2 is an Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) early gene in the ie1-ie2 gene region for which there is no homolog in either the archetype virus, Autographa californica MNPV, or Bombyx mori NPV. opep-2 is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early gene ie1, which produces spliced mRNAs. However, distinct from ie1, the multiple mRNAs of opep-2 are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8-12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene an opep-2 deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells.

  1. Tissue culture of ornamental cacti

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    Eugenio Pérez-Molphe-Balch


    Full Text Available Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

  2. Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures (United States)

    Singh, Ratnesh K.; Mallela, Ramya K.; Cornuet, Pamela K.; Reifler, Aaron N.; Chervenak, Andrew P.; West, Michael D.; Wong, Kwoon Y.; Nasonkin, Igor O.


    Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for

  3. Characterization and Clinical Implication of Th1/Th2/Th17 Cytokines Produced from Three-Dimensionally Cultured Tumor Tissues Resected from Breast Cancer Patients

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    Anna Kiyomi


    Full Text Available OBJECTIVES: Several cytokines secreted from breast cancer tissues are suggested to be related to disease prognosis. We examined Th1/Th2/Th17 cytokines produced from three-dimensionally cultured breast cancer tissues and related them with patient clinical profiles. METHODS: 21 tumor tissues and 9 normal tissues surgically resected from breast cancer patients were cultured in thermoreversible gelatin polymer–containing medium. Tissue growth and Th1/Th2/Th17 cytokine concentrations in the culture medium were analyzed and were related with hormone receptor expressions and patient clinical profiles. RESULTS: IL-6 and IL-10 were expressed highly in culture medium of both cancer and normal tissues. However, IFN-γ, TNF-α, IL-2, and IL-17A were not detected in the supernatant of the three-dimensionally cultured normal mammary gland and are seemed to be specific to breast cancer tissues. The growth abilities of hormone receptor–negative cancer tissues were significantly higher than those of receptor-positive tissues (P = 0.0383. Cancer tissues of stage ≥IIB patients expressed significantly higher TNF-α levels as compared with those of patients with stage tissues resected from breast cancer patients can grow in the three-dimensional thermoreversible gelatin polymer culture system and produce Th1/Th2/Th17 cytokines. Hormone receptor–positive cancer tissues showed less growth ability. TNF-α is suggested to be a biomarker for the cancer stage.

  4. Tissue culture in forestry and agriculture

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    Henke, R.R.; Hughes, K.W.; Constantin, M.J.; Hollaender, A. (eds.)


    This symposium aims to examine the current state-of-the-art in tissue culture technology and to relate this state of technology to practical, applied, and commercial interests. Thus the focus is on embryogenesis in culture: how to recognize it, factors which affect embryogenesis, use of embryogenic systems, etc.; and variability from culture. A special session on woody species again emphasized somatic embryogenesis as a means of rapid propagation. This volume emphasizes tissue culture of forest trees.

  5. Plant Tissue Culture in a Bag. (United States)

    Beck, Mike


    Describes the use of an oven bag as a sterile chamber for culture initiation and tissue transfer. Plant tissue culture is an ideal tool for introducing students to plants, cloning, and experimental design. Includes materials, methods, discussion, and conclusion sections. (SAH)

  6. Addressing the Instability of DNA Nanostructures in Tissue Culture


    Hahn, Jaeseung; Wickham, Shelley F. J.; Shih, William M.; Perrault, Steven D.


    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify...

  7. Characterization of eight terpenoids from tissue cultures of the Chinese herbal plant, Tripterygium wilfordii, by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. (United States)

    Su, Ping; Cheng, Qiqing; Wang, Xiujuan; Cheng, Xiaoqing; Zhang, Meng; Tong, Yuru; Li, Fei; Gao, Wei; Huang, Luqi


    In this study, a reliable method for analysis and identification of eight terpenoids in tissue cultures of Tripterygium wilfordii has been established using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS). Our study indicated that sterile seedlings, callus cultures and cell-suspension cultures can rapidly increase the amount of biological materials. HPLC-ESI-MS was used to identify terpenoids from the extracts of these tissue cultures. Triptolide, triptophenolide, celastrol and wilforlide A were unambiguously determined by comparing the retention times, UV spectral data, and mass fragmentation behaviors with those of the reference compounds. Another four compounds were tentatively identified as triptonoterpenol, triptonoterpene, 22β-hydroxy-3-oxoolean-12-en-29-oic acid and wilforlide B, based on their UV and mass spectrometry spectra. The quantitative analysis showed that all three materials contain triptolide, triptophenolide, celastrol, wilforlide A, and the contents of the four compounds in the cell-suspension cultures were 53.1, 240, 129 and 964 µg/g, respectively, which were at least 2.0-fold higher than these in the sterile seedlings and callus cultures. Considering the known pharmacological activity of triptolide and celastrol, we recommend the cell-suspension cultures as biological materials for future studies, such as clinical and toxicological studies. The developed method was validated by the evaluation of its precision, linearity, detection limits and recovery, and it was successfully used to identify and quantify the terpenoids in the tissue cultures.

  8. Progress in planta transformation without tissue culture

    Institute of Scientific and Technical Information of China (English)

    GU Yun-Hong; YU Zeng-Liang; QIN Guang-Yong; HUO Yu-Ping


    With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly.

  9. Cultivation of Hepatitis Virus in Tissue Culture. (United States)

    as the feeder source and primary human hepatocytes as the feeder layer. Feeder layers are also being formed using other sensitive tissue culture lines including human diploid lung cells (WI 38) and Hela cells .


    Institute of Scientific and Technical Information of China (English)

    成少飞; 叶清


    Objective To evaluate the feasibility of utilizing vascular cells combined with folded and framed culture model to develop completely autologous human tissue without using any scaffold material under the principles of Tissue Engineering. Methods Human vascular cells cultured from ascending aorta (group A) and saphenous vein (group B) were seeded into 15cm-dishes (each n =12 ) and cultured to form cell sheets over a period of four weeks with Dulbecco's modified Eagle's medium supplemented with lmmol/L L-ascorbic acid 2-phosphate. Thereafter, cell sheets (6 samples of each group) were four-layer folded and cultured in a newly developed frame device for additional four weeks. Controls remained under standard culture conditions. Tissue development was evaluated by light and electron microscopy, biochemical assays. Results The formation of multi-layered cell sheets and production of extracellular matrix were observed in each group after the initial four weeks. Analysis of the folded and framed neo-tissue revealed a solid structure with increased matrix formation and tissue organization compared to the control groups after additional four weeks. DNA assay indicated significantly lower cell proliferation in folded and framed cell sheets than in that of unframed counterparts. Yet hydroxyproline assay demonstrated significant increase of collagen content in the framed aortic and venous derived tissues, which contained 82 % and 42 % that of human pericardium. Conclusion It is feasible to obtain completely autologous human cardiovascular tissue with the alternative new approach. Numerous issues including improvement of mechanical strength of neo-tissue remain to be investingated.

  11. The autologus graft of epithelial tissue culture

    Directory of Open Access Journals (Sweden)

    Minaee B


    Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

  12. Substituted Indoleacetic Acids Tested in Tissue Cultures

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen


    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable...

  13. Production and Characterization of a Novel, Electrospun, Tri-Layer Polycaprolactone Membrane for the Segregated Co-Culture of Bone and Soft Tissue

    Directory of Open Access Journals (Sweden)

    Sasima Puwanun


    Full Text Available Composite tissue-engineered constructs combining bone and soft tissue have applications in regenerative medicine, particularly dentistry. This study generated a tri-layer, electrospun, poly-ε-caprolactone membrane, with two microfiber layers separated by a layer of nanofibers, for the spatially segregated culture of mesenchymal progenitor cells (MPCs and fibroblasts. The two cell types were seeded on either side, and cell proliferation and spatial organization were investigated over several weeks. Calcium deposition by MPCs was detected using xylenol orange (XO and the separation between fibroblasts and the calcified matrix was visualized by confocal laser scanning microscopy. SEM confirmed that the scaffold consisted of two layers of micron-diameter fibers with a thin layer of nano-diameter fibers in-between. Complete separation of cell types was maintained and calcified matrix was observed on only one side of the membrane. This novel tri-layer membrane is capable of supporting the formation of a bilayer of calcified and non-calcified connective tissue.

  14. 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...

  15. Development of germ-free plants and tissue culture (United States)

    Venketeswaran, S.


    The botanical program is reported for experiments performed at the Lunar Receiving Laboratory. Papers prepared during this program are listed. The studies reported include: tissues cultured on various mediums, nutritional studies, preparation of plant cultures for Apollo 15, and pine tissue cultures.

  16. 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.

  17. 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

  18. 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.

  19. Study Progress on Tissue Culture of Maize Mature Embryo (United States)

    Wang, Hongzhen; Cheng, Jun; Cheng, Yanping; Zhou, Xioafu

    It has been paid more and more attention on maize tissue culture as it is a basic work in maize genetic transformation, especially huge breakthrough has been made in maize tissue culture utilizing mature embryos as explants in the recent years. This paper reviewed the study progress on maize tissue culture and plant regeneration utilizing mature embryos as explants from callus induction, subculture, plant regeneration and browning reduction and so on.

  20. Trends in the use of tissue culture in forest improvement

    Energy Technology Data Exchange (ETDEWEB)

    Haissig, B.E.; Nelson, N.D.; Kidd, G.H.


    We have analyzed and described the problems and potentials of using tissue culture in micropropagation and biotechnologies related to forest improvement. Trends in forest management concepts, commerical micropropagation, and tissue culture biotechnologies are discussed. Our analysis suggests that tissue culture will contribute significantly to the improvement of forests through exploitation of existing genotypes and production of new, commercially valuble genotypes. Such changes may significantly influence worldwide management decisions in forestry. 97 references.

  1. Multispectral tissue characterization for intestinal anastomosis optimization (United States)

    Cha, Jaepyeong; Shademan, Azad; Le, Hanh N. D.; Decker, Ryan; Kim, Peter C. W.; Kang, Jin U.; Krieger, Axel


    Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement.

  2. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten;


    Free-floating roller tube (FFRT) cultures of fetal rat and human nigral tissue are a means for tissue storage prior to grafting in experimental Parkinson's disease. In the present study, FFRT cultures prepared from embryonic-day-14 rat ventral mesencephalon were maintained for 4, 8, 12, or 16 days...

  3. Addressing the instability of DNA nanostructures in tissue culture. (United States)

    Hahn, Jaeseung; Wickham, Shelley F J; Shih, William M; Perrault, Steven D


    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable

  4. Addressing the Instability of DNA Nanostructures in Tissue Culture (United States)


    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg2+-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg2+ to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable experimental

  5. Effect of lunar materials on plant tissue culture. (United States)

    Walkinshaw, C. H.; Venketeswaran, S.; Baur, P. S.; Croley, T. E.; Scholes, V. E.; Weete, J. D.; Halliwell, R. S.; Hall, R. H.


    Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials.

  6. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon


    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  7. Alkaloid production by callous tissue cultures of Cereus peruvianus (Cactaceae). (United States)

    de Oliveira, Arildo José Braz; Machado, Maria Fátima Pires da Silva


    The morphologically undifferentiated cells of nonregenerant callous tissue of Cereus peruvianus cultured in the original medium and in medium supplemented with tyrosine were used as an alkaloid source. Comparison of alkaloid production by C. peruvianus plants and by callous tissues indicated that alkaloid levels were almost twice as high in callous tissues as in shoots of C. peruvianus plants. The ratio of alkaloid concentration between mature plant and morphologically undifferentiated cells of callous tissue was 1:1.7. A relationship between culture medium containing tyrosine and alkaloid production was also observed in the callous tissues of C. peruvianus. Since increased alkaloid production may be induced by additional factors such as tyrosine, increasing levels of tyrosine or other conditions of the culture medium may be considered factors for inducing higher alkaloid production by C. peruvianus callous tissues.

  8. Study of cultured bovine capsular bag in pure ocular tissue

    Institute of Scientific and Technical Information of China (English)

    WANG Yan-qing; LI Qiu-ming


    @@ The proliferation, differentiation and fibrosis of lens epithelia cells (LECs) is mainly responsible for posterior capsular opacification (PCO). From the primary culture of LECs to the culture of lens capsular bag, the models of posterior capsular opacification have been developed. At present, the most commonly used model is cell culture in medium with serum. But the culture in pure ocular tissue has not been reported. Therefore, we established a new model of posterior capsular opacification-culturing bovine lens capsular bag in pure ocular tissue to exclude the role of serum. Our study established a new culture method to investigate the proliferation,differentiation and apoptosis of lens epithelia cells in the aqueous humor with or without lens cortex and vitreous humor. The purpose of the study is to model posterior capsular opacification in vivo as closely as possible and to discuss the influence of ocular tissue on posterior capsular opacification.

  9. 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)

  10. 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...

  11. Co-culture in cartilage tissue engineering

    NARCIS (Netherlands)

    Hendriks, Jeanine; Riesle, Jens; Blitterswijk, van Clemens A.


    For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to ob

  12. Tissue-Culture Method of Cloning Rubber Plants (United States)

    Ball, E. A.


    Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

  13. 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.

  14. [Issues of large scale tissue culture of medicinal plant]. (United States)

    Lv, Dong-Mei; Yuan, Yuan; Zhan, Zhi-Lai


    In order to increase the yield and quality of the medicinal plant and enhance the competitive power of industry of medicinal plant in our country, this paper analyzed the status, problem and countermeasure of the tissue culture of medicinal plant on large scale. Although the biotechnology is one of the most efficient and promising means in production of medicinal plant, it still has problems such as stability of the material, safety of the transgenic medicinal plant and optimization of cultured condition. Establishing perfect evaluation system according to the characteristic of the medicinal plant is the key measures to assure the sustainable development of the tissue culture of medicinal plant on large scale.

  15. Ultrasound Tissue Characterization of Vulnerable Atherosclerotic Plaque

    Directory of Open Access Journals (Sweden)

    Eugenio Picano


    Full Text Available A thrombotic occlusion of the vessel fed by ruptured coronary atherosclerotic plaque may result in unstable angina, myocardial infarction or death, whereas embolization from a plaque in carotid arteries may result in transient ischemic attack or stroke. The atherosclerotic plaque prone to such clinical events is termed high-risk or vulnerable plaque, and its identification in humans before it becomes symptomatic has been elusive to date. Ultrasonic tissue characterization of the atherosclerotic plaque is possible with different techniques—such as vascular, transesophageal, and intravascular ultrasound—on a variety of arterial segments, including carotid, aorta, and coronary districts. The image analysis can be based on visual, video-densitometric or radiofrequency methods and identifies three distinct textural patterns: hypo-echoic (corresponding to lipid- and hemorrhage-rich plaque, iso- or moderately hyper-echoic (fibrotic or fibro-fatty plaque, and markedly hyperechoic with shadowing (calcific plaque. Hypoechoic or dishomogeneous plaques, with spotty microcalcification and large plaque burden, with plaque neovascularization and surface irregularities by contrast-enhanced ultrasound, are more prone to clinical complications than hyperechoic, extensively calcified, homogeneous plaques with limited plaque burden, smooth luminal plaque surface and absence of neovascularization. Plaque ultrasound morphology is important, along with plaque geometry, in determining the atherosclerotic prognostic burden in the individual patient. New quantitative methods beyond backscatter (to include speed of sound, attenuation, strain, temperature, and high order statistics are under development to evaluate vascular tissues. Although not yet ready for widespread clinical use, tissue characterization is listed by the American Society of Echocardiography roadmap to 2020 as one of the most promising fields of application in cardiovascular ultrasound imaging

  16. Stability of Propofol in Polystyrene-Based Tissue Culture Plates (United States)

    Sall, Jeffrey W.; Leong, Jason


    Propofol has been reported to have high stability in glass and relatively high stability up to 24 hours in polyvinyl chloride-based medical plastics. Recent publications have observed the effects of propofol on cells and tissues grown in culture. Many cell culture plastics are formulated from polystyrene but we could find little information on the stability of propofol exposed to these products. We observed very little change in the concentration of propofol diluted in cell culture medium over 24 hours when exposed to glass, but substantial loss of the drug when exposed to 96-well polystyrene cell culture plates. This decrease was most rapid in the first hour but continued until 24 hours. The type of plastic used in cell and tissue culture experiments with propofol may influence the results by increasing the apparent dose required to see an effect. PMID:23632056

  17. Use of diathermy for weeding heterogeneous tissue cultures. (United States)

    Marks, R M; Penny, R


    Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelial cells. Primary cell cultures were observed at X 100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture.

  18. Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture. (United States)

    AbouZid, S


    Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture.

  19. Selection of bacterial wilt-resistant tomato through tissue culture. (United States)

    Toyoda, H; Shimizu, K; Chatani, K; Kita, N; Matsuda, Y; Ouchi, S


    Bacterial wilt-resistant plants were obtained using a tomato tissue culture system. A virulent strain ofPseudomonas solanacearum secreted some toxic substances into the culture medium. Leaf explant-derived callus tissues which were resistant to these toxic substances in the culture filtrate were selectedin vitro and regenerated into plants. These plants expressed bacterial wilt resistance at the early infection stage to suppress or delay the growth of the inoculated bacteria. On the other hand, complete resistance was obtained in self-pollinated progeny of regenerants derived from non-selected callus tissues. These plants showed a high resistance when inoculated with this strain, and were also resistant when planted in a field infested with a different strain of the pathogen.

  20. Using Data Fusion to Characterize Breast Tissue

    Energy Technology Data Exchange (ETDEWEB)

    Littrup, P; Duric, N; Leach, R R; Azevedo, S G; Candy, J V; Moore, T; Chambers, D H; Mast, J E; Johnson, S A; Holsapple, E


    New ultrasound data, obtained with a circular experimental scanner, are compared with data obtained with standard X-ray CT. Ultrasound data obtained by scanning fixed breast tissue were used to generate images of sound speed and reflectivity. The ultrasound images exhibit approximately 1 mm resolution and about 20 dB of dynamic range. All data were obtained in a circular geometry. X-ray CT scans were used to generate X-ray images corresponding to the same 'slices' obtained with the ultrasound scanner. The good match of sensitivity, resolution and angular coverage between the ultrasound and X-ray data makes possible a direct comparison of the three types of images. We present the results of such a comparison for an excised breast fixed in formalin. The results are presented visually using various types of data fusion. A general correspondence between the sound speed, reflectivity and X-ray morphologies is found. The degree to which data fusion can help characterize tissue is assessed by examining the quantitative correlations between the ultrasound and X-ray images.

  1. The role of silicon in plant tissue culture. (United States)

    Sivanesan, Iyyakkannu; Park, Se Won


    Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

  2. 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...

  3. The use of animal tissues alongside human tissue: Cultural and ethical considerations. (United States)

    Kaw, Anu; Jones, D Gareth; Zhang, Ming


    Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display.

  4. Adaptive image segmentation applied to plant reproduction by tissue culture (United States)

    Vazquez Rueda, Martin G.; Hahn, Federico; Zapata, Jose L.


    This paper presents that experimental results obtained on indoor tissue culture using the adaptive image segmentation system. The performance of the adaptive technique is contrasted with different non-adaptive techniques commonly used in the computer vision field to demonstrate the improvement provided by the adaptive image segmentation system.

  5. [Tissue culture of medicinal plant and abscisic acid]. (United States)

    Fang, Hui-Yong; Zhu, Hong; Yao, Jian-Xun; Jia, Cai-Feng; Shan, Gao-Wei; Li, Min-Hui


    Abscisic acid (ABA) plays a key role in many physiological processes of plants, and it was also applied to fields of medicinal plant biotechnology. The article presents a review of some recent application of ABA in enhancing the production of secondary metabolites of medicinal plants, improving the in vitro conservation in medicinal plant tissue culture system.

  6. Preparation of crude rough microsomes from tissue culture cells. (United States)

    Sabatini, David D


    There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

  7. Practical Instruction in Tissue Culture and Cytogenetics for Sandwich Students. (United States)

    Williams, D. C.; Bishun, N. P.


    Describes the training and practical techniques taught to students involved in a sandwich course at the Tissue Culture and Cytogenetics Unit of the Marie Curie Memorial Foundation, Surrey, England. Students spend a minimum of six months involved in the sandwich course before returning to university for a final academic year. (JR)

  8. The role of activated charcoal in plant tissue culture. (United States)

    Thomas, T Dennis


    Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

  9. Characterization of mesenchymal stem cells derived from equine adipose tissue

    Directory of Open Access Journals (Sweden)

    A.M. Carvalho


    Full Text Available Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs in horses through (1 the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2 flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O, osteogenic (Alizarin Red, and chondrogenic (Alcian Blue. The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.

  10. Systems biology characterization of engineered tissues. (United States)

    Rajagopalan, Padmavathy; Kasif, Simon; Murali, T M


    Tissue engineering and molecular systems biology are inherently interdisciplinary fields that have been developed independently so far. In this review, we first provide a brief introduction to tissue engineering and to molecular systems biology. Next, we highlight some prominent applications of systems biology techniques in tissue engineering. Finally, we outline research directions that can successfully blend these two fields. Through these examples, we propose that experimental and computational advances in molecular systems biology can lead to predictive models of bioengineered tissues that enhance our understanding of bioengineered systems. In turn, the unique challenges posed by tissue engineering will usher in new experimental techniques and computational advances in systems biology.

  11. [Asepsis sowing and tissue culture of Bletilla striata]. (United States)

    Zeng, Songjun; Huang, Xiangli; Chen, Zhilin; Chen, Jiantong; Duan, Jun


    The asepsis sowing and tissue culture of Bletilla striata were studied. The results indicated that the embyro culture had highest sprouting percent and plantlets percent when their embryos were mature. The optimal medium for the embryo culture was 1/2 MS. Adding 10% coconut juice can promote embyros sprouting and plantlets formation, 1% active carbon can improve plantlets growing. The best medium for the top of plantlet on culture and multiplication was 1/2 MS + 6-BA 0.5 mg/L + NAA 0.2 mg/L. The best medium of rooting was 1/2 MS + NAA 0.5 mg/L, and 10% banana juice can improve rooting of plantlets.

  12. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  13. Research on Rapid Propagation of Gongshui Pomelo by Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    Shuqiong WU; Kaiming SHI; Yanfang ZHU; Zhiyuan LI; Qian MOU


    Objective This study aimed to investigate the optimal medium and hor-mone combinations for efficient rapid propagation of Gongshui pomelo and analyze key technical measures in the tissue culture process. [Method] Stem tips and stem segments with buds were col ected from four varieties of pomelo adult trees as ex-plants, to investigate the main effect and key regulatory factors of vegetative organs and tissue culture explants and to propose a series of measures to prevent and control microbial contamination. Final y, an efficient rapid propagation technology system of Gongshui pomelo was established. [Result] Spring shoot explants con-tained large amounts of auxin, cytokinins, gibberel ins and other growth regulators, which could be used for tissue culture with high bud generation rate and rapid growth. Different conditions led to various culture results. Specifical y, mature pomelo seeds should be generated on semi-solid 1/2MS medium and transferred to solid MS medium for incubation. The propagation coefficient of stem segments with axil-lary buds was greater than that of stem tips, exhibiting significant differences. In ad-dition, the optimal hormone combination was 6-BA 0.5 mg/L + NAA 0.5 mg/L, which significantly promoted the induction and differentiation of adventitious buds. [Conclu-sion] This study provided basis for basic research, production and application of pomelo germplasm resources.

  14. Determinants of microstructural load transfer in cartilage tissue from chondrocyte culture (United States)

    Fedewa, Michelle Marie


    The goals of this research were to (i) develop a tissue model system for studying the microstructure of matrix produced by chondrocytes, (ii) characterize the biochemical and mechanical properties of the chondrocyte culture tissue, (iii) evaluate the response of the chondrocyte culture tissue to various stimulants (retinoic acid, interleukin-1beta, and xyloside), (iv) investigate the roles of proteoglycan and collagen in the tearing and tensile properties of a chondrocyte culture tissue, and (v) develop a finite element model of the chondrocyte culture tissue microstructure to study its tensile pre-failure properties. The roles of proteoglycan and collagen were explored by experimentation using a cultured cartilage tissue, and by development of a theoretical finite element model which related the cartilage tissue microstructure to its macroscopic properties. Tear and tensile testing was performed. Failure testing is valuable because it is known that cracks exist and propagate from the cartilage surface in osteoarthritic joints. It was found that collagen was important for providing the material stiffness of the cultured tissue, and that both collagen and proteoglycan were important for providing the tear toughness of the tissue. It was also found that as the collagen density or collagen material stiffness increased, the material stiffness of the cultured tissue increased, and as the proteoglycan or collagen densities increased, the tear toughness of the tissue increased. A three-dimensional finite element microstructural model of cartilage was developed, consisting of linear elastic collagen fibrils embedded in a linear viscoelastic proteoglycan solid matrix. Fluid flow in the cartilage matrix was not included in this model. Viscoelastic time dependent behavior was an appropriate model for the cartilage. The results of this model were comparable to the experimental results, as well as to past continuum models of cartilage. Collagen and proteoglycan material moduli

  15. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

    Directory of Open Access Journals (Sweden)

    Trisha N. Peel


    Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

  16. Characterization of tendon cell cultures of the human rotator cuff. (United States)

    Pauly, S; Klatte, F; Strobel, C; Schmidmaier, G; Greiner, S; Scheibel, M; Wildemann, B


    Rotator cuff tears are common soft tissue injuries of the musculoskeletal system that heal by formation of repair tissue and may lead to high retear rates and joint dysfunction. In particular, tissue from chronic, large tendon tears is of such degenerative nature that it may be prone to retear after surgical repair. Besides several biomechanical approaches, biologically based strategies such as application of growth factors may be promising for increasing cell activity and production of extracellular tendon matrix at the tendon-to-bone unit. As a precondition for subsequent experimental growth factor application, the aim of the present study was to establish and characterize a human rotator cuff tendon cell culture. Long head biceps (LHB)- and supraspinatus muscle (SSP)- tendon samples from donor patients undergoing shoulder surgery were cultivated and examined at the RNA level for expression of collagen type-I, -II and -III, biglycan, decorin, tenascin-C, aggrecan, osteocalcin, tenomodulin and scleraxis (by Real-time PCR). Finally, results were compared to chondrocytes and osteoblasts as control cells. An expression pattern was found which may reflect a human rotator cuff tenocyte-like cell culture. Both SSP and LHB tenocyte-like cells differed from chondrocyte cell cultures in terms of reduced expression of collagen type-II (ptendon matrix and osteofibroblastic integration at the tendon-bone unit following tendon repair.

  17. Mineralization and growth of cultured embryonic skeletal tissue in microgravity (United States)

    Klement, B. J.; Spooner, B. S.


    Microgravity provides a unique environment in which to study normal and pathological phenomenon. Very few studies have been done to examine the effects of microgravity on developing skeletal tissue such as growth plate formation and maintenance, elongation of bone primordia, or the mineralization of growth plate cartilage. Embryonic mouse premetatarsal triads were cultured on three space shuttle flights to study cartilage growth, differentiation, and mineralization, in a microgravity environment. The premetatarsal triads that were cultured in microgravity all formed cartilage rods and grew in length. However, the premetatarsal cartilage rods cultured in microgravity grew less in length than the ground control cartilage rods. Terminal chondrocyte differentiation also occurred during culture in microgravity, as well as in the ground controls, and the matrix around the hypertrophied chondrocytes was capable of mineralizing in both groups. The same percentage of premetatarsals mineralized in the microgravity cultures as mineralized in the ground control cultures. In addition, the sizes of the mineralized areas between the two groups were very similar. However, the amount of 45Ca incorporated into the mineralized areas was significantly lower in the microgravity cultures, suggesting that the composition or density of the mineralized regions was compromised in microgravity. There was no significant difference in the amount of 45Ca liberated from prelabeled explants in microgravity or in the ground controls.

  18. Commercializing plant tissue culture processes: economics, problems and prospects

    Energy Technology Data Exchange (ETDEWEB)

    Sahai, O.; Knuth, M.


    Novel tissue culture techniques and a range of process schemes may be considered for commercial production of plant derived drugs, chemicals, flavors and cosmetics. Plant cell immobilization, in conjunction with strain selection and product leakage, represents a major technological advancement, with significant economic implications. Conventional batch processes produce high value products at low production capacities, whereas continuous biocatalytic processes can potentially enable production of plant derived chemicals in the $20-$25/kg price range.

  19. Early fruit setting from tissue culture-derived mangosteen tree

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato


    Full Text Available Vitro-plantlets of mangosteen derived from culturing young leaves were acclimatized in 1993. Small and large polybag seedlings were carefully raised under controlled environmental conditions until 1994 when they were ready to be transferred to the field. During this stage, morphological abnormalities of the seedlings were recorded. After transferring to the field for 5-6 years (1994-1999 at Yi Ngo District, Narathiwat Province and Klong Hoi Khong District, Songkhla Province, morphological characters of the plants were again observed in comparison with seed-derived plants. The results showed that tissue culture-derived plants were more bushy and started blooming 5 years after planting while the seed-derived plants still had tall canopy (not bushy and were not bearing fruit in the same period of time. However, the blooming of cultured plants did not give the fruit setting in the first blooming year. All flowers dropped off completely. Heavy fruit setting was observed in the following year (2000. Tissue culture trees had smaller but healthier leaves whereas seed-derived trees had pale yellowish green leaves. Fruit qualities in terms of total soluble solids (TSS and total acids (TA were not much different between the two types of these mangosteen trees.

  20. Tissue characterization by using narrow band imaging (United States)

    Gono, Kazuhiro


    NBI (Narrow Band Imaging) was first introduced in the market in 2005 as a technique enabling to enhance image contrast of capillaries on a mucosal surface(1). It is classified as an Optical-Digital Method for Image-Enhanced Endoscopy(2). To date, the application has widely spread not only to gastrointestinal fields such as esophagus, stomach and colon but also the organs such as bronchus and bladder. The main target tissue of NBI enhancement is capillaries. However, findings of many clinical studies conducted by endoscopy physicians have revealed that NBI observation enables to enhance more other structures in addition to capillaries. There is a close relationship between those enhanced structures and histological microstructure of a tissue. This report introduces the tissue microstructures enhanced by NBI and discusses the possibility of optimized illumination wavelength in observing living tissues.

  1. Characterizing the elastic properties of tissues

    Directory of Open Access Journals (Sweden)

    Riaz Akhtar


    Full Text Available The quality of life of ageing populations is increasingly determined by age-related changes to the mechanical properties of numerous biological tissues. Degradation and mechanical failure of these tissues has a profound effect on human morbidity and mortality. Soft tissues have complex and intricate structures and, similar to engineering materials, their mechanical properties are controlled by their microstructure. Thus age-relate changes in mechanical behavior are determined by changes in the properties and relative quantities of microstructural tissue components. This review focuses on the cardiovascular system; it discusses the techniques used both in vivo and ex vivo to determine the age-related changes in the mechanical properties of arteries.

  2. Characterization of tendon cell cultures of the human rotator cuff

    Directory of Open Access Journals (Sweden)

    S Pauly


    Full Text Available tator cuff tears are common soft tissue injuries of the musculoskeletal system that heal by formation of repair tissue and may lead to high retear rates and joint dysfunction. In particular, tissue from chronic, large tendon tears is of such degenerative nature that it may be prone to retear after surgical repair. Besides several biomechanical approaches, biologically based strategies such as application of growth factors may be promising for increasing cell activity and production of extracellular tendon matrix at the tendon-to-bone unit. As a precondition for subsequent experimental growth factor application, the aim of the present study was to establish and characterize a human rotator cuff tendon cell culture.Long head biceps (LHB- and supraspinatus muscle (SSP- tendon samples from donor patients undergoing shoulder surgery were cultivated and examined at the RNA level for expression of collagen type-I, -II and -III, biglycan, decorin, tenascin-C, aggrecan, osteocalcin, tenomodulin and scleraxis (by Real-time PCR. Finally, results were compared to chondrocytes and osteoblasts as control cells.An expression pattern was found which may reflect a human rotator cuff tenocyte-like cell culture. Both SSP and LHB tenocyte-like cells differed from chondrocyte cell cultures in terms of reduced expression of collagen type-II (p≤0.05 and decorin while higher levels of collagen type-I were seen (p≤0.05. With respect to osteoblasts, tenocyte-like cells expressed lower levels of osteocalcin (p≤0.05 as well as tenascin C, biglycan and collagen type-III. Expression of scleraxis, tenomodulin and aggrecan was similar between all cell types.This study represents a characterization of tenocyte-like cells from the human rotator cuff as close as possible. It helps analyzing their biological properties and allows further studies to improve production of tendon matrix and osteofibroblastic integration at the tendon-bone unit following tendon repair.

  3. Tissue culture of three species of Laurencia complex (United States)

    Shen, Songdong; Wu, Xunjian; Yan, Binlun; He, Lihong


    To establish a micropropagation system of three Laurencia complex species ( Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus) by tissue culture techniques, we studied the regeneration characteristics and optimal culture conditions of axenic algal fragments cultured on solid medium and in liquid medium. Regeneration structures were observed and counted regularly under a reverse microscope to investigate the regeneration process, polarity and optimal illumination, and temperature and salinity levels. The results show that in most cultures of the three species, we obtained bud regeneration on solidified medium with 0.5% agar and in liquid medium. Rhizoid-like regeneration was filamentous and developed from the lower cut surface of fragments in L. okamurai, but was discoid and developed from the apical back side of bud regeneration in L. tristicha and C. undulatus. Regeneration polarity was localized to the apical part of algal fronds in all three species, and on fragments cut from the basal part of algae buds could develop from both the upper and the lower cut surfaces. Buds could develop from both the medullary and the cortical portions in L. okamurai and C. undulatus, while in L. tristicha, buds only emerged from the cortex. The optimal culture conditions for L. okamurai were 4 500 lx, 20°C and 35 (salinity); for C. undulatus, 4 500 lx, 20°C and 30; and for L. tristicha, 4 500 lx, 25°C and 30.

  4. Organoid culture systems for prostate epithelial and cancer tissue. (United States)

    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans


    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material contain the differentiated luminal and basal cell types, whereas organoids derived from prostate cancer tissue mimic the histology of the tumor. We explain how to establish these cultures in the fully defined serum-free conditioned medium that is required to sustain organoid growth. Starting with the plating of digested tissue material, full-grown organoids can usually be obtained in ∼2 weeks. The culture protocol we describe here is currently the only one that allows the growth of both the luminal and basal prostatic epithelial lineages, as well as the growth of advanced prostate cancers. Organoids established using this protocol can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis and drug discovery.

  5. Tissue Culture and Rapid Multiplication Techniques of Apocynum L.

    Institute of Scientific and Technical Information of China (English)


    [Objective] This study aimed to investigate rapid multiplication of Apocynum by tissue culture so as to provide plantlet sources for its industrialized cultivation. [Method] The asepsis seedlings were obtained by dealing with Apocynum seeds. Its cotyledons, hypocotyls and shoot tips were cultured on the media containing different concentrations of hormones. Finally, the influence of different hormone combinations on differentiation of cotyledons and hypocotyls, rapid multiplication of shoot tips, rapid multiplication of regenerated shoots, and rooting of test-tube plantlets was com- pared. [Result] MS+2.0 mg/L BA+0.03 mg/L NAA and MS+0.07 mg/L NAA were the optimum medium for inducing regenerated buds from cotyledons and hypocotyls re- spectively; MS+2.0 mg/L BA+0.02 mg/L NAA was the best medium for rapid multi- plication of shoot tips; MS+1.9 mg/L BA+I.7 mg/L NAA was the best medium for rapid multiplication of regenerated buds: and 1/2MS+0.6 mg/L NAA was the best medium for inducing roots. [Conclusion] The optimum hormone combination was de- termined for Apocynum rapid multiplication by tissue culture, which provides technical support on Apocynum industrialized cultivation.

  6. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components. (United States)


    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and components. (a) Identification. Synthetic... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media...

  7. Shoot tissue culture of Robinia pseudoacacia f. decaisneana

    Institute of Scientific and Technical Information of China (English)


    Robinia pseudoacacia f. decaisneana is a transfiguration of Robinia pseudoacacia. For enhancing propagation coefficient of the species, the experiment of shoot tissue culture of Robinia pseudoacacia f. decaisneana was conducted in Forestry College of Shenyang Agricultural University from July 1999 to July 2001. The experiment included medium selection of explant induction survival, initial culture, subculture as well as rooting culture, and forming seedling with callus. The results showed that shoot segment in vitro survive rate is larger in spring than in autumn, and green dense callus could form plantlet. The best medium for initial culture was SH+0.5mg/L BA+0.05 mg/L NAA, with a propagation coefficient of 4.1 (per micro-cutting in a month), and for subculture it was B5+0.5 mg/L BA+0.05 mg/L NAA+ 10 mg/L Glu., with a propagation coefficient of 4.7. The best rooting medium was 1/2MS+0.5 mg/L NAA+10 mg/L Glu., with a rooting rate of 84.4%. These results provide reference data for reproduction of superior individuals of Robinia pseudoacacia f. decaisneana.

  8. Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

    Directory of Open Access Journals (Sweden)

    Urszula Adamczyk-Rogozińska


    Full Text Available The conditions for the regeneration of plants through organogenesis from callus tissues of Menyanthes trifoliata are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M containing indole-3-acetic acid (IAA 0,5 mg/l and 6-benzyladenine (BA 1 mg/l or zeatin (2 mg/l. Under these conditions ca 7 shoots (mostly 1 cm or more in length per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

  9. Entropy analysis of OCT signal for automatic tissue characterization (United States)

    Wang, Yahui; Qiu, Yi; Zaki, Farzana; Xu, Yiqing; Hubbi, Basil; Belfield, Kevin D.; Liu, Xuan


    Optical coherence tomography (OCT) signal can provide microscopic characterization of biological tissue and assist clinical decision making in real-time. However, raw OCT data is noisy and complicated. It is challenging to extract information that is directly related to the pathological status of tissue through visual inspection on huge volume of OCT signal streaming from the high speed OCT engine. Therefore, it is critical to discover concise, comprehensible information from massive OCT data through novel strategies for signal analysis. In this study, we perform Shannon entropy analysis on OCT signal for automatic tissue characterization, which can be applied in intraoperative tumor margin delineation for surgical excision of cancer. The principle of this technique is based on the fact that normal tissue is usually more structured with higher entropy value, compared to pathological tissue such as cancer tissue. In this study, we develop high-speed software based on graphic processing units (GPU) for real-time entropy analysis of OCT signal.

  10. Contamination and browning in tissue culture of Platanus occidentalis L.

    Institute of Scientific and Technical Information of China (English)

    Tao Feng-jie; Zhang Zhi-yi; Zhou Jun; Yao Na; Wang Dong-mei


    Twigs of 2-3-year-old Platanus occidentalis L. were used as experimental material to find the causes for the contamination and browning in the initial stages of tissue cultures. To compare the degree of browning of explants picked off from different growing seasons,the experimental material was excised from trees on each of the first ten days in January, March,May and July,2006. The results indicated that the contamination and browning rates of the material cut off in January (14. 2% and 30. 6%. respectively)and March were somewhat lower than those in July. The pretreatment of soaking the explants in different anti-oxidants and absorbents at the same time could diminish some side effects. The pretreatment of using 10 g·L-1 vitamin C reduced the contamination and browning rate effectively. An orthogonal experiment showed that the optimal factor and level arrangement is 0. 5 mg·L-1 BA,2. 0 g·L-1 active carbon and 1. 5 g·L-1 PVP which resulted in a browning rate of only 16. 5%. In general,sampling period,physical properties and pretreatment of explants are the main factors responsible for the contamination and browning of material in the initial stages of P. occidentalis tissue cultures.

  11. Characterization of cell cultures in contact with different orthopedic implants biomaterials (United States)

    Ouenzerfi, G.; Hannoun, A.; Hassler, M.; Brizuela, L.; Youjil, S.; Bougault, C.; Trunfio-Sfarghiu, A.-M.


    The aim of this study is to identify the role of biological and mechanical constraints (at the cellular level) surrounding living tissues (cartilage and bone) in the presence of different joint implant biomaterials. In this fact, cells cultures in the presence of different types of biomaterials (pyrolytic carbon, cobalt-Chromium, titanium) has been performed. These cell cultures were subjected to biological characterization tests and mechanical characterization. The obtained results correlate with the in vivo observations (a promotion of the creation of a neocartilagical tissue in contact with the Pyrolytic Carbon implants).

  12. Characterizing Tissue with Acoustic Parameters Derived from Ultrasound Data

    Energy Technology Data Exchange (ETDEWEB)

    Littrup, P; Duric, N; Leach, R R; Azevedo, S G; Candy, J V; Moore, T; Chambers, D H; Mast, J E; Johnson, S A; Holsapple, E


    In contrast to standard reflection ultrasound (US), transmission US holds the promise of more thorough tissue characterization by generating quantitative acoustic parameters. We compare results from a conventional US scanner with data acquired using an experimental circular scanner operating at frequencies of 0.3 - 1.5 MHz. Data were obtained on phantoms and a normal, formalin-fixed, excised breast. Both reflection and transmission-based algorithms were used to generate images of reflectivity, sound speed and attenuation.. Images of the phantoms demonstrate the ability to detect sub-mm features and quantify acoustic properties such as sound speed and attenuation. The human breast specimen showed full field evaluation, improved penetration and tissue definition. Comparison with conventional US indicates the potential for better margin definition and acoustic characterization of masses, particularly in the complex scattering environments of human breast tissue. The use of morphology, in the context of reflectivity, sound speed and attenuation, for characterizing tissue, is discussed.

  13. Characterizing tissue with acoustic parameters derived from ultrasound data (United States)

    Littrup, Peter J.; Duric, Nebojsa; Leach, Richard, Jr.; Azevedo, Steve G.; Candy, James V.; Moore, Thomas; Chambers, David H.; Mast, Jeffrey E.; Holsapple, Earle


    In contrast to standard reflection ultrasound (US), transmission US holds the promise of more thorough tissue characterization by generating quantitative acoustic parameters. We compare results from a conventional US scanner with data acquired using an experimental circular scanner operating at frequencies of 0.3 - 1.5 MHz. Data were obtained on phantoms and a normal, formalin-fixed, excised breast. Both reflection and transmission-based algorithms were used to generate images of reflectivity, sound speed and attenuation.. Images of the phantoms demonstrate the ability to detect sub-mm features and quantify acoustic properties such as sound speed and attenuation. The human breast specimen showed full field evaluation, improved penetration and tissue definition. Comparison with conventional US indicates the potential for better margin definition and acoustic characterization of masses, particularly in the complex scattering environments of human breast tissue. The use of morphology, in the context of reflectivity, sound speed and attenuation, for characterizing tissue, is discussed.

  14. Homoeologous gene silencing in tissue cultured wheat callus

    Directory of Open Access Journals (Sweden)

    Chapman Natalie H


    Full Text Available Abstract Background In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses. Results Evidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR single strand conformation polymorphism (SSCP platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested. Conclusion Our results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.

  15. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. (United States)

    Li, Xingnan; Nadauld, Lincoln; Ootani, Akifumi; Corney, David C; Pai, Reetesh K; Gevaert, Olivier; Cantrell, Michael A; Rack, Paul G; Neal, James T; Chan, Carol W-M; Yeung, Trevor; Gong, Xue; Yuan, Jenny; Wilhelmy, Julie; Robine, Sylvie; Attardi, Laura D; Plevritis, Sylvia K; Hung, Kenneth E; Chen, Chang-Zheng; Ji, Hanlee P; Kuo, Calvin J


    The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

  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. Application of tissue characterization in intravascular ultrasound imaging (United States)

    Mullen, William L.; Fitzgerald, Peter J.; Yock, Paul G.


    Current intravascular ultrasound imaging technology is able to determine the extent and distribution of pathologic processes within the vessel wall, but is not highly sensitive in discriminating between certain types of tissue. `Tissue characterization' refers to a set of computer-based techniques that utilize features of the ultrasound signal beyond basic amplitude to help define the composition of the tissue of interest. This technique involves quantitative analysis of the ultrasound signals reflected from tissue before these signals pass through the processing steps in the ultrasound instrument.

  18. Thermal property of biological tissues characterized by piezoelectric photoacoustic technique

    Institute of Scientific and Technical Information of China (English)

    GAO Chunming; ZHANG Shuyi; CHEN Yan; SHUI Xiuji; YANG Yuetao


    A photoacoustic piezoelectric method based on a simplified thermoelastic theory is employed to determine thermal diffusivities of biological tissues. The thermal diffusivities of porcine tissues with different preparation conditions, including fresh, dry and specially prepared conditions, are characterized. Comparing the experimental evaluated diffusivities of the tissues in three conditions with each other, it can be seen that the diffusivities of the fresh tissues are the biggest and the diffusivities of the specially prepared tissues are bigger than that of the dry ones generally. The results show that the piezoelectric photoacoustic method is especially effective for determining macro-effective (average) thermal diffusivities of biological materials with micro- inhomogeneity and easy to be performed, which can provide useful information for researching thermal characters of biological tissues.


    Directory of Open Access Journals (Sweden)

    Dagmara Kullačová


    Full Text Available Orobanchaceae family includes parasitic plants that attack many important food crops. Genus Phelipanche, belonging to this family is considered to cause high negative impact on food production. Developing Phelipanche plant must establish connection with the root of host plant, from which it receives all resources needed for further development. Nowadays big effort is directed to finding a reliable strategy to control parasitic plants. In vitro cultures of P. ramosa can be genetically manipulated and used for study of genes involved in host-parasite interactions. We established in vitro cultures of parasitic species Phelipanche ramosa on solid and liquid media in parallel. The obtained results point out that development of P. ramosa calli was origin specific. We tested the effect of antibiotic kanamycin on in vitro cultures of Phelipanche ramosa with aim to develop system for its genetic manipulation and selection of transgenic tissue using kanamycin- resistance approach. The selection pressure of kanamycin was stronger in liquid grown cultures. However, concentrations of kanamycin tested (up to 250 mg.l-1 did not ensured elimination of kanamycine non-resistant tissue. Tests of other candidate selection markers are currently in progress.

  20. Study of Nosocomial Clostridium Difficile Diarrhea by Culture and Tissue Culture Methods

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    MH Salari


    Full Text Available Background: Clostridium difficult is an identified cause of antibiotic-associated diarrhea, antibiotic-associated colitis, pseudomembranous colitis and nosocomial diarrhea. The purpose of this study was to investigate the prevalence of nosocomial C. difficile-associated diarrhea in Tehran University of Medical Science Hospitals. Methods: In this study a total of 942 stool samples from patients with nosocomial diarrhea that were hospitalized in Imam Khomeini hospital, Shariati hospital and Children clinical center were collected. The samples were cultured on a selective cycloserine cefoxitin fructose agar (CCFA and incubated in anaerobic conditions, at 37 °C for 5 days. Isolated C. difficile by conventional biochemical tests, bacterial cytotoxicity by Vero tissue culture and antimicrobial sensitivity to antibiotics by Kirby Bauer method (disk diffusion were investigated. Results: Of the total patients, 57 Toxigenic C. difficile (6.1% were isolated. Results of statistical analysis show significant differences between the rate of isolated Toxigenic C. difficile and age group of patients (P< 0.05. Among the units of selected hospitals, Toxigenic C. difficile was isolated most frequently in gastroenterology of Children clinical center. Meanwhile, the isolated Toxigenic C. difficile were sensitive to vancomycin, Chloramphenicol and ceftriaxone. Conclusion: Our findings show that, Toxigenic C. difficile was found in 6.1% hospitalized patients. Therefore, further studies to evaluate the role of Toxigenic C. difficile in nosocomial diarrhea processes, ecological and pathogenic terms by culture, Tissue culture and molecular methodes are suggested.

  1. Micropropagation and maintenance of phytoplasmas in tissue culture. (United States)

    Bertaccini, Assunta; Paltrinieri, Samanta; Martini, Marta; Tedeschi, Mara; Contaldo, Nicoletta


    Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

  2. Application of tissue culture technology to southern forestry

    Energy Technology Data Exchange (ETDEWEB)

    Brown, C. L.

    Much progress has been made during the past few years in cloning difficult to propagate conifers and hardwoods. The predicted rate of progress during the next decade will increase several fold because of the increased interest and number of researchers now engaged in tissue culture research in America and in various laboratories around the world. The immediate application to forestry practice will depend on developing operable systems of cloning valuable plus tree selections by the thousands in laboratory flasks followed by transferring these plantlets to preformed tublings containing necessary nutrients and mycorrhizal fungi for rapid development in semi-controlled greenhouses prior to field planting. Laboratory screening for mutants, new hybrid combinations, and haploid lines, will be used more and more in the future to develop improved strains or lines of forest trees. The use of these various techniques accompanied by shorter forest rotations will significantly increase the yields of fiber and cellulose in the South within the next couple of decades.

  3. Tissue culture and micropropagation for forest biomass production

    Energy Technology Data Exchange (ETDEWEB)

    Mason, E.; Maine, F.W.


    An increase in forest production will be necessary in the future when wood becomes a major renewable source of energy and chemicals along with its traditional role of fibre source. This increase could eventually by achieved be proper selection and breeding of trees. Clonal forestry by vegetative propagation of cuttings is becoming a viable alternative to a seedling-based forestry with many advantages, and cutting could be used to quickly propagate large numbers of clones of control-pollinated seedlings. Most forest trees are propagated sexually and seed orchards were started in the US and Canada in the last 40-50 years for breeding purposes. Forests could ultimately be established with improved seedlings instead of from seed with unknown genetic potential, or by natural regeneration. Micropropagation is the term used to refer to the propagation of plants raised by tissue culture methods rather than from seeds or cuttings. Many clonal plantlets could be regenerated asexually in the laboratory and eventually transplanted to permanent sites. In addition the technology could be developed to produce new variants from somatic cells. Tissue culture is a technique which may be useful for plant propagation where conventional methods are inadequate or unsuitable. However, traditional studies of field planting observed over long periods of time would still be necessary. This document has the object of informing those who may wish to know more about these techniques in relation to practical application, and require a general overview rather than experimental details, which are given in an annotated bilbiography. 274 refs., 2 figs., 1 tab.

  4. Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles. (United States)

    Daquinag, Alexes C; Souza, Glauco R; Kolonin, Mikhail G


    White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet

  5. Organotypic culture of human bone marrow adipose tissue. (United States)

    Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji


    The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

  6. Delivery of recombinant alphavirus into hippocampal slice tissue culture. (United States)

    Lundstrom, Kenneth


    The alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have shown reduced cytotoxicity and prolonged expression. This protocol describes gene delivery of recombinant alphavirus to hippocampal slice cultures. Organotypic slices are covered by a layer of glial cells that impedes the penetration of viral particles to the neurons. Thus, viral particles should be injected manually into the extracellular space of the tissue.


    Directory of Open Access Journals (Sweden)

    Cuiqin Yang


    Full Text Available The tissue culture and rapid propagation of Cleome spinosa Jacq. was explored by investigating the effects of different plant growth regulators on callus induction, bud differentiation, and root formation of three types of explants. The results showed that hypocotyls and stem segments regenerated buds directly on growth regulator-free Murashige and Skoog (MSmedium. The highest callus induction rates of hypocotyls, stem segments, and leaves reached 100% and were obtained on the culture medium of MS + (1.0 to 2.0 mg/L kinetin (KT + 0.02 mg/L α-naphthalene acetic acid (NAA, on which the leaves produced the best quality of calluses. The leaf-derived calluses were subcultured on MS + 0.5 mg/L KT +0.5 mg/L 6-benzylaminopurine (BAP and achieved the highest differentiation rate of 100%, producing an average of 7.5 buds per explant. Inoculation with MS + 0.5 mg/L indole-3-butyric acid (IBA resulted in the production of a number of thick roots by 66.7% of the regenerated buds. After transplanting, plantlets with more roots survived easily and grew well.

  8. Plant Generation of TM-1 via Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    LIU Chuan-liang; LI Fu-guang; XU Leng-chun


    @@ Plant generation of TM-1 via tissue culture was established.The hypocotyledon sections as explants which were cultured in a series of improved MS media containing 0.05~0.10 mg · L-1 IAA, 0.1~0.15 mg · L-1 Kt,0.07~0.14 mg · L-1 2,4-D could produce a large number of calli which were easier to regeneration in this system.The calli,which were subcuhured in another MS media containing 0.03~0.05 mg · L-1 Kt for 3-4 times produced embryoid callus in a rate of 35%.Fifty-six somatic embryoid calli were subcultured in an improved MS medium containing 0.1 mg·L-1 BA and 0.1~0.15 mg · L-1 IAA for plant regenerating,and 47 cotton plantlets were regenerated from them.

  9. Biotransformation of tissue-specific hormone tibolone with fungal culture Trichothecium roseum (United States)

    Shah, Syed Adnan Ali; Sultan, Sadia; Zaimi bin Mohd Noor, M.


    Whole cells based biotransformation is an important tool for bioconversion of steroids. It can be used to synthesize biologically potent compounds with diverse structures. Biotransformation of tissue-specific hormone tibolone (1) with Trichothecium roseum (ATCC 13411) has being carried out for the first time. Two new and three known metabolites 2-6 were isolated from fermentation of tibolone (1) with Trichothecium roseum and their structures were characterized by 2D NMR spectroscopy and mass spectrometry. The relative stereochemistry of new metabolites 5 and 6 was deduced by 2D NOESY experiments. The effect of cultures on tibolone structural modifications and time-course studies has also been conducted.

  10. Characterization of European Management Perspective Hofstede's Cultural Dimensions Comparative Analysis

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    Full Text Available Because Europe is characterized by the coexistence of several cultures whose characteristics have both similarities and differences but appreciable, the results of researchers in this regard are different from each other, this distinction is often made based on the prevailing values of that culture , which determines the orientation of the country for a certain system, management style or to a specific profile manager. A particularly important role in characterizing cultural factors play European management, each differing from the other culture as module in addressing various fundamental issues that characterize that society. These issues can be characterized by certain general cultural dimensions that Hofstede defines them as aspects of a culture that can be measured in relation to other cultures. The differences between management systems in European countries (mainly EU countries in the context of this article, the study is based on four cultural dimensions of Hofstede model (power distance, individualism vs. collectivism, uncertainty avoidance, femininity vs. masculinity and change scores recorded for these dimensions in each country. Dimensions considered primarily affect organizational culture which in turn significantly influence the development and performance of the organization and its members, management practices and policies.Data from Hofstede's study reinforce and support the claim that European countries can be grouped systematically cultural groups (Nordic countries, Latin, Germanic, Anglo-Saxon, Eastern Europe that allow significant interpretation in terms of management organization, and that can speak of a typical single European culture but you can see all dimensions of cultural differences taken into account.

  11. Characterizing parameters of Jatropha curcas cell cultures for microgravity studies (United States)

    Vendrame, Wagner A.; Pinares, Ania


    Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters

  12. Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. (United States)

    Carrier, R L; Papadaki, M; Rupnick, M; Schoen, F J; Bursac, N; Langer, R; Freed, L E; Vunjak-Novakovic, G


    Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.

  13. 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.

  14. Adipose-Derived Mesenchymal Stromal/Stem Cells: Tissue Localization, Characterization, and Heterogeneity

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    Patrick C. Baer


    Full Text Available Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared to other sources. It is easily accessible in large quantities with minimal invasive harvesting procedure, and isolation of adipose-derived mesenchymal stromal/stem cells (ASCs yields a high amount of stem cells, which is essential for stem-cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in a perivascular niche, whereas the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to plastic. Nevertheless, recent isolation and culture techniques lack standardization. Cultured cells are characterized by their expression of characteristic markers and their capacity to differentiate into cells from meso-, ecto-, and entodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, neural cells, and endothelial and epithelial cells. Nevertheless, recent studies suggest that ASCs are a heterogeneous mixture of cells containing subpopulations of stem and more committed progenitor cells. This paper summarizes and discusses the current knowledge of the tissue localization of ASCs in situ, their characterization and heterogeneity in vitro, and the lack of standardization in isolation and culture methods.

  15. 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

  16. Characterization of growth hormone and prolactin produced by human pituitary in culture. (United States)

    Skyler, J S; Rogol, A D; Lovenberg, W; Knazek, R A


    Fragments of a pituitary tumor from a patient with acromegaly were grown in tissue culture. The tumor secreted both growth hormone and prolactin,which were recovered in high concentrations. The nonpurified hormones were characterized and compared to their respective counterparts obtained by extraction from normal pituitaries obtained at autopsy. The tissue culture and pituitary extracted hormones were eluted from Sephadex G-100 with the same partition coefficients. Growth hormone from both sources showed parallel dose-response displacement curves, by logit-log transformation, in both specific immunoassay and in a specific lymphocyte binding assay. Prolactin from both sources was compared in specific immunoassay using three different antisera. Parallel logit-log displacement curves were seen with one antiserum, while the other two antisera yielded non-parallel curves, indicating structural differences between prolactin from the two sources. Quantitative polyacrylamide gel electrophoresis was performed using multiphasic buffer systems previously developed for characterization of each hormone. By the criteria of joint 95% confidence envelopes of retardation co-efficient and relative free mobility, tissue culture growth hormone and prolactin were indistinguishable from their pituitary-extracted counterparts. This study demonstrates that, prior to purification, tissue culture derived hormone can be characterized by multiple criteria and compared to a standard preparation. Structural differences can be detected, as in the case of prolactin. When the hormones are indistinguishable, as in the case of growth hormone, it becomes worthwhile to increase the scale of tissue cultured production, with the prospect that tissue culture may serve as a source of hormone for both experimental and therapeutic use.

  17. Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles. (United States)

    Merz, Lea; Höbel, Sabrina; Kallendrusch, Sonja; Ewe, Alexander; Bechmann, Ingo; Franke, Heike; Merz, Felicitas; Aigner, Achim


    The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350μm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of

  18. An electrochemical approach to monitor pH change in agar media during plant tissue culture. (United States)

    Wang, Min; Ha, Yang


    In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.

  19. Investigation of Various Tissue Culture Monolayers Sensitivity in Detection of Clostridium difficile Toxin

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    MH Salari


    Full Text Available Backround: Clostridium difficile is the most common cause of nosocomial diarrhea. It is usually a consequence of antibi­otic treatment, but sporadic cases can occur. The purpose of this study was to investigate five tissue culture monolayers sen­sitivity in detection of C. difficile-toxin. Methods: A total of 402 stool samples from patients with nosocomial diarrhea hospitalized in three hospitals of Tehran Uni­versity of Medical Sciences (TUMS were collected. The samples were cultured on a selective cycloserine cefoxitin fructose agar (CCFA and incubated in anaerobic conditions, at 37 °C for 4 days. Isolates were characterized to species level by con­ventional biochemical tests. Bacterial cytotoxicity was assayed on five tissue culture monolayers. Results: Our findings show that of the total patients, 24 toxigenic C. difficile (6% were isolated. All 24 C. difficile toxins showed cytotoxic effect at ³ 1:10 dilution on Hela, Hep2, Vero, McCoy and Mdck cells after 16, 20, 24, 24 and 30 hours, re­spectively. C. difficile toxin showed cytotoxic effect at ³ 1:100 dilutions only on Hela cell monolayer after 48 hours. Conclusion: Hela cell monolayer may be a satisfactory substitute for the detection of C. difficile toxin in clinical specimens.   

  20. Cloning of medicinal plants through tissue culture--a review. (United States)

    Chaturvedi, H C; Jain, Madhu; Kidwai, N R


    In order to have standardized formulations, the chemical constituents from plants and their parts are required to be uniform both qualitatively and quantitatively. Furthermore, an ever increasing demand of uniform medicinal plants based medicines warrants their mass cloning through plant tissue culture strategy. A good number of medicinal plants have been reported to regenerate in vitro from their various parts, but a critical evaluation of such reports reveals that only a few complete medicinal plants have been regenerated and still fewer have actually been grown in soil, while their micropropagation on a mass scale has rarely been achieved, particularly in those medicinal plants where conventional propagation is inadequate, like, the mass clonal propagation of Dioscorea floribunda leading to its successful field trials. Such facts make it imperative to document the factual position of micropropagation of medicinal plants bringing out the advancements made along with the short falls, in this important area. The present review deals with the futuristic view on the said subject restricted to higher plants.

  1. Embryogenesis and plant regeneration of Medicago spp. in tissue culture. (United States)

    Nagarajan, P; McKenzie, J S; Walton, P D


    Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2-4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced.

  2. Image-based characterization of foamed polymeric tissue scaffolds

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    Mather, Melissa L; Morgan, Stephen P; Crowe, John A [School of Electrical and Electronic Engineering, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); White, Lisa J; Shakesheff, Kevin M [School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Tai, Hongyun; Howdle, Steven M [School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom); Kockenberger, Walter [School of Physics and Astronomy, University of Nottingham, University Park, Nottingham NG7 2RD (United Kingdom)], E-mail:


    Tissue scaffolds are integral to many regenerative medicine therapies, providing suitable environments for tissue regeneration. In order to assess their suitability, methods to routinely and reproducibly characterize scaffolds are needed. Scaffold structures are typically complex, and thus their characterization is far from trivial. The work presented in this paper is centred on the application of the principles of scaffold characterization outlined in guidelines developed by ASTM International. Specifically, this work demonstrates the capabilities of different imaging modalities and analysis techniques used to characterize scaffolds fabricated from poly(lactic-co-glycolic acid) using supercritical carbon dioxide. Three structurally different scaffolds were used. The scaffolds were imaged using: scanning electron microscopy, micro x-ray computed tomography, magnetic resonance imaging and terahertz pulsed imaging. In each case two-dimensional images were obtained from which scaffold properties were determined using image processing. The findings of this work highlight how the chosen imaging modality and image-processing technique can influence the results of scaffold characterization. It is concluded that in order to obtain useful results from image-based scaffold characterization, an imaging methodology providing sufficient contrast and resolution must be used along with robust image segmentation methods to allow intercomparison of results.

  3. A Generalized Gamma Mixture Model for Ultrasonic Tissue Characterization

    Directory of Open Access Journals (Sweden)

    Gonzalo Vegas-Sanchez-Ferrero


    Full Text Available Several statistical models have been proposed in the literature to describe the behavior of speckles. Among them, the Nakagami distribution has proven to very accurately characterize the speckle behavior in tissues. However, it fails when describing the heavier tails caused by the impulsive response of a speckle. The Generalized Gamma (GG distribution (which also generalizes the Nakagami distribution was proposed to overcome these limitations. Despite the advantages of the distribution in terms of goodness of fitting, its main drawback is the lack of a closed-form maximum likelihood (ML estimates. Thus, the calculation of its parameters becomes difficult and not attractive. In this work, we propose (1 a simple but robust methodology to estimate the ML parameters of GG distributions and (2 a Generalized Gama Mixture Model (GGMM. These mixture models are of great value in ultrasound imaging when the received signal is characterized by a different nature of tissues. We show that a better speckle characterization is achieved when using GG and GGMM rather than other state-of-the-art distributions and mixture models. Results showed the better performance of the GG distribution in characterizing the speckle of blood and myocardial tissue in ultrasonic images.

  4. Advances in tissue engineering through stem cell-based co-culture. (United States)

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


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

  5. Measuring NO Production by Plant Tissues and Suspension Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Jan Vitecek; Vilem Reinohl; Russell L.Jones


    We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants.The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap.The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors.We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources.NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected,as was the production of NO from nitrite by the incubation medium of barley aleurone.Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up.We could also measure NO production from elicitor-treated suspension cultured cells using this set-up.Further,we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues.In addition to the dramatic effects that pH can have on fluorescence from DAF-FM,the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results.Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

  6. Collagen gels and the 'Bornstein legacy': from a substrate for tissue culture to cell culture systems and biomaterials for tissue regeneration. (United States)

    García-Gareta, Elena


    As collagen is the main structural component of connective tissues and skin, much effort was made in the past and still today to use it in cell culture applications. Moreover, collagen biomaterials are widely used in tissue regeneration, including the treatment of burns and chronic wounds. The great implications of the research carried out by Bornstein, Ehrmann and Gey on collagen preparations in the 1950s for cell culture and more recently tissue engineering and regeneration are described in this commentary. Specifically, it is explored why the 1958 paper on 'Reconstituted Rat-Tail Collagen Used as Substrate for Tissue Cultures on Coverslips in Maximow Slides and Roller Tubes' by M. B. Bornstein has made an invaluable contribution to the field.

  7. Comparative study on Allium schoenoprasum cultivated plant and Allium schoenoprasum tissue culture organs antioxidant status. (United States)

    Stajner, D; Popović, B M; Calić-Dragosavac, D; Malenčić, D; Zdravković-Korać, S


    This study was designed to examine Allium schoenoprasum tissue culture organs antioxidant and scavenging activity and to make a comparison between Allium schoenoprasum cultivated plant and Allium schoenoprasum tissue culture organs antioxidant activity. This study reports the results on the root, stalk and leaf antioxidant enzyme activities (superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase), reduced glutathione quantity, flavonoids and soluble protein contents and quantities of malonyldialdehyde and ·OH radical. In Allium schoenoprasum tissue culture organs the total antioxidant capacity was determined by the FRAP method and scavenger activity by the DPPH method. The present results indicated that the crude extract of Allium schoenoprasum tissue culture exhibited antioxidant and scavenging abilities in all investigated plant parts, especially in the roots. According to our results, the tissue culture plants exhibited the highest activities in the roots in contrast to the cultivated plants where highest activities were observed in the leaves.

  8. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes

    DEFF Research Database (Denmark)

    Du, Zhen-Yu; Ma, Tao; Lock, Erik-Jan;


    We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal...... levels of IL-6, TNF-a and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar...... in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited...

  9. Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture. (United States)

    Masuta, Yukari; Nozawa, Kosuke; Takagi, Hiroki; Yaegashi, Hiroki; Tanaka, Keisuke; Ito, Tasuku; Saito, Hideyuki; Kobayashi, Hisato; Matsunaga, Wataru; Masuda, Seiji; Kato, Atsushi; Ito, Hidetaka


    A transposition of a heat-activated retrotransposon named ONSEN required compromise of a small RNA-mediated epigenetic regulation that includes RNA-directed DNA methylation (RdDM) machinery after heat treatment. In the current study, we analyzed the transcriptional and transpositional activation of ONSEN to better understand the underlying molecular mechanism involved in the maintenance and/or induction of transposon activation in plant tissue culture. We found the transposition of heat-primed ONSEN during tissue culture independently of RdDM mutation. The heat activation of ONSEN transcripts was not significantly up-regulated in tissue culture compared with that in heat-stressed seedlings, indicating that the transposition of ONSEN was regulated independently of the transcript level. RdDM-related genes were up-regulated by heat stress in both tissue culture and seedlings. The level of DNA methylation of ONSEN did not show any change in tissue culture, and the amount of ONSEN-derived small RNAs was not affected by heat stress. The results indicated that the transposition of ONSEN was regulated by an alternative mechanism in addition to the RdDM-mediated epigenetic regulation in tissue culture. We applied the tissue culture-induced transposition of ONSEN to Japanese radish, an important breeding species of the family Brassicaceae. Several new insertions were detected in a regenerated plant derived from heat-stressed tissues and its self-fertilized progeny, revealing the possibility of molecular breeding without genetic modification.

  10. Efficient biomarkers for the characterization of bone tissue. (United States)

    Gil, J E; Aranda, J P; Mérida-Casermeiro, E; Ujaldón, M


    This work describes an expert system aimed to an accurate classification of cell tissue on microscopic images coming from studies of bone tissue regeneration from stem cells. We analyze a wide number of phenotype and color issues to build effective vectors of features for the subsequent characterization of tissue into five different classes: bone, cartilage, muscle, fiber and spine. The features selection includes texture, shape and color descriptors, among which we consider color histograms, Zernike moments and circular parameters. Once a preliminary set of vectors candidates are selected, several trained and non-parametric classifiers based on neural networks, decision trees, Bayesian classifiers and association rules are analyzed, and later compared with unsupervised methods to determine those that fit more closely to our needs for distinguishing bone tissue. Because of the high resolution of our biomedical images, we effectively decompose them into smaller windows for a faster execution, with the impact of the window size being discussed in terms of speed and robustness. Our final study compares accuracy and computational time together with different stainings for revealing tissue properties: Picrosirius red, alcian blue and safranin blue. Overall, safranin blue reveals as the best staining and multilayer perceptron as the most effective classifier.

  11. Propagation of jarrah (Eucalyptus marginata) by organ and tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, M.J.; McComb, J.A.


    Micropropagation methods are described for the production of clonal lines from Eucalyptus marginata (jarrah) seedlings. Nodal explants from mature trees can also yield shoot cultures, but a high frequency of contamination occurs among such explants. Uncontaminated callus cultures can be produced from mature trees by culturing stamen filaments and shoots can subsequently be regenerated from this callus. The rooting percentage of shoot cultures from either nodes or stamen callus of mature trees is low compared with that from seedling explants. Considerable variation was observed between trees in the ability of stamen callus to regenerate shoots and in the frequency of rooting. (Refs. 27)

  12. Automatic 3D acoustic tissue models from histologic tissue sections and application to ex vivo tissue characterization (United States)

    Mamou, Jonathan; Oelze, Michael L.; O'Brien, William D.; Zachary, James F.


    Three-dimensional acoustic tissue models (3DATMs) can be used as computational tools for ultrasonic imaging algorithm development and analysis. 3DATMs are automatically constructed from digitized light microscope images of consecutive H&E-stained histologic tissue sections. Construction necessitated contrast equalization, registration, and interpolation of missing sections. The registered (with interpolated) sections yield a 3D histologic volume (3DHV). Acoustic properties are then assigned to each tissue constituent of the 3DHV to obtain the 3DATM. A tissue characterization technique was developed to obtain scatterer parameter estimates (size and acoustic concentration) from a 3D impedance map (3DZM) deduced from a 3DHV by assigning acoustic impedance values. 3DZMs were constructed for a rat fibroadenoma (FA), a mouse mammary tumor (MMT) and a mouse sarcoma (EHS). From these 3 3DZMs estimates, effective scatterer diameters of 91 μm, 31.5 μm, and 34.5 μm, respectively, were determined. Independent ultrasonic measurements yielded average scatterer diameters of 105 μm, 30 μm, and 33 μm, respectively. The 3DZM estimation scheme showed results similar to those obtained by the ultrasonic measurements. 3DATMs may therefore be a useful tool for quantifying ultrasonic tissue properties. [Work supported by the University of Illinois Research Board.

  13. Performance evaluation of ultrasonic Nakagami image in tissue characterization. (United States)

    Tsui, Po-Hsiang; Yeh, Chih-Kuang; Chang, Chien-Cheng; Chen, Wen-Shiang


    Conventional ultrasonic B-mode images qualitatively describe tissue structures but are unsuitable for quantitative analyses of scatterer properties. We have recently developed an ultrasonic parametric imaging technique based on the Nakagami statistical distribution that is able to quantify scatterer concentrations. The aim of the present study is to further explore both the behavior of a Nakagami image in characterizing different scatterer structures at different signal-to-noise ratios (SNRs) and the feasibility of Nakagami imaging using a general commercial ultrasound scanner for tissue examinations. Simulations, experiments on a tissue-mimicking phantom and in vitro measurements on a muscle tissue before and after microwave treatment were carried out. The SNR and contrast-to-noise ratio (CNR) were estimated to quantify image performance. The results demonstrate that a Nakagami image can differentiate different scatterer concentrations for single, hypoechoic and hyperechoic targets. Also, a Nakagami image, when combined with an ultrasound scanner, can complement the B-scan to characterize tissue and to identify the region of interest with a larger CNR. However, the noise effect can degrade the performance of a Nakagami image. When the signal SNR decreased to 15 dB in simulations and to 8 dB in experiments, the CNR of the hyperechoic Nakagami image decreased by 4% and 27%, respectively, and that of the hypoechoic one decreased by 42% and 80%, respectively. These results indicate that a Nakagami image behaves well in identifying regions with high scatterer concentrations but does not perform well when both the scatterer concentration and SNR are low.

  14. Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

    Energy Technology Data Exchange (ETDEWEB)

    Sluis, C.


    The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media and rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.


    Abstract: Palatal organ culture provides an in vitro model for the study of the formation of the secondary palate, which forms the roof of the mouth in the developing fetus. The protocol describes the steps for culture of the mid-facial region of the fetal mouse or rat. In cult...

  16. Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue.

    Directory of Open Access Journals (Sweden)

    Yinzhi Lai

    Full Text Available It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG were cultured on two dimensional (2D flat surfaces and in three dimensional (3D synthetic poly-L-lactic acid (PLLA and polystyrene (PS polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K(+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells' functionality, transcriptase expression and related membrane protein distributions (caveolin-1 were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.

  17. Efforts to accelerate domestication of winged bean (Psophocarpus tetragonolobus (L.) DC.) by means of induced mutations and tissue culture.

    NARCIS (Netherlands)

    Klu, G.Y.P.


    This thesis describes mutation breeding and tissue culture techniques developed for accelerated domestication of winged bean ( Psophocarpustetragonolobus (L.) DC.). The tissue culture techniques, which are the first steps towards genetic transformation of the crop, include: (1) direct adventitious s

  18. Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor (United States)

    Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.


    PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

  19. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues. (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu


    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  20. Tissue Characterization on Ultrasound Harmonic Signals using Nakagami Statistics (United States)

    Lin, Fanglue; Cristea, Anca; Cachard, Christian; Basset, Olivier

    Quantitative ultrasound (QUS) imaging provides a way to characterize biological tissue. The QUS estimates can be obtained from the envelope statistics. Previous studies are mainly based on the whole backscattered signals analysis. However, the ultrasound propagation is a nonlinear process and the harmonic signals can therefore reveal the nonlinear nature of a biological medium. The present study investigates the statistics of harmonic signal envelopes to relate the distribution parameters to the nonlinear coefficients. The main results demonstrate that the distributions exhibit a different behavior for fundamental and harmonic signals and that media with different nonlinearities can be distinguished, when using Nakagami statistics on the harmonic signal envelopes.

  1. Tissue Characterization on Common Carotid Artery using AR Model (United States)

    Koya, Yoshiharu; Mizoshiri, Isao

    This study uses one image with a echocardiography and detects arteriosclerosis on the common carotid artery by tissue characterization. The conventional methods are measurement of thickness on the vessel wall or stiffness on the common carotid artery. However, they need frames of several beats and great calculation times. But, we detect the arteriosclerosis with only one image. Our method estimate the grade of arteriosclerosis by AR model parameters fitted to the texture on the lumen-intima wall. Experimental results show the validity of the proposed method.

  2. Therapeutically important proteins from in vitro plant tissue culture systems. (United States)

    Doran, Pauline M


    Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.

  3. Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue. (United States)

    Varettas, Kerry


    As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006-2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO2, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.

  4. Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants. (United States)

    Rasool, Rafia; Ganai, Bashir Ahmad; Akbar, Seema; Kamili, Azra Nahaid; Dar, Muhammad Younus; Masood, Akbar


    Wild and tissue culture raised regenerants of Artemisia amygdalina, a critically endangered and endemic plant of Kashmir and North West Frontier Provinces of Pakistan were screened for the amount of bioactive principles and in particular antimalarial compound artemesinin. Phytochemical screening of extracts revealed the presence of terpenes, alkaloids, phenolics, tannins (polyphenolics), cardiac glycosides and steroids in wild (aerial, inflorescence) and tissue culture regenerants (in vitro grown plant, callus and green house acclimatized plants). HPLC of Artemisia amygdalina revealed the presence of artemesinin in petroleum ether extracts of wild aerial part, tissue culture raised plant and green house acclimatized plants. Acetonitrile and water in 70:30 ratios at flow rate of 1ml/min was standardised as mobile phase. Retention time for standard chromatogram was 6.7. Wild inflorescences and callus does not produce artemesinin. This is the first report of phytochemical screening and artemesinin estimation of wild and tissue culture raised regenerants of Artemisia amygdalina.

  5. How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture. (United States)

    Haldeman, Janice H.; Ellis, Jane P.


    Presents techniques used for disinfestation of plant material, preparation of equipment and media, and laboratory procedures for tissue culture using cauliflower. Details methods for preparing solutions and plant propagation by cloning. (CW)

  6. An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues. (United States)

    Li, Xingnan; Ootani, Akifumi; Kuo, Calvin


    Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

  7. In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation. (United States)

    Shih, Sharon M-H; Doran, Pauline M


    Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82+/-0.14 mg g(-1) dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80-90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 microg g(-1) dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.

  8. Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors. (United States)

    Saegeman, V; Verhaegen, J; Lismont, D; Verduyckt, B; De Rijdt, T; Ectors, N


    Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

  9. Study on the propagation of some sugar cane clones derived from tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Chagvardieff, P.; Mauboussin, J.C.; Weil, J.


    The study of the propagation by cuttings of sugar-cane clones derived from tissue cultures showed that the emergence of one bud setts was variable. A definition of parameters for this germination is proposed: germination vigour, germination capacity, shooting vigour, shooting capacity. Variants optained, among them germination or shooting vigour, were always improved when compared with the control; this might result from a cell rejuvenation induced by the tissue culture. 11 references.

  10. Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

    Directory of Open Access Journals (Sweden)

    KM Elson


    Full Text Available Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis, camptothecin (to induce apoptosis or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH, alkaline phosphatase (AP, glycosaminoglycans (GAGs, and matrix metalloproteinase (MMP-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

  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. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons. (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C


    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  13. A High Rate Tension Device for Characterizing Brain Tissue

    CERN Document Server

    Rashid, Badar; Gilchrist, Michael; 10.1177/1754337112436900


    The mechanical characterization of brain tissue at high loading velocities is vital for understanding and modeling Traumatic Brain Injury (TBI). The most severe form of TBI is diffuse axonal injury (DAI) which involves damage to individual nerve cells (neurons). DAI in animals and humans occurs at strains > 10% and strain rates > 10/s. The mechanical properties of brain tissues at these strains and strain rates are of particular significance, as they can be used in finite element human head models to accurately predict brain injuries under different impact conditions. Existing conventional tensile testing machines can only achieve maximum loading velocities of 500 mm/min, whereas the Kolsky bar apparatus is more suitable for strain rates > 100/s. In this study, a custom-designed high rate tension device is developed and calibrated to estimate the mechanical properties of brain tissue in tension at strain rates < 90/s, while maintaining a uniform velocity. The range of strain can also be extended to 100% de...

  14. Isolation and characterization of a monoclonal anti-protein kinase CK2 beta-subunit antibody of the IgG class for the direct detection of CK2 beta-subunit in tissue cultures of various mammalian species and human tumors

    DEFF Research Database (Denmark)

    Nastainczyk, W; Schmidt-Spaniol, I; Boldyreff, B;


    -subunit or in the CK2 holoenzyme (alpha 2 beta 2). Here, concentrations of the first antibody of 1 ng/ml still allowed the detection of the subunit. Immunoblotting of crude cellular extracts from various tissue cultures (man, mouse, and hamster), from human tumors, and the nonneoplastic tissue allowed the detection...... of the CK2 beta-subunit. The detected epitope of this antibody was, as determined by the epitope analysis technique, 123GLSDI127....

  15. Tissue Culture Responses from Different Explants of Rice

    Institute of Scientific and Technical Information of China (English)

    WANG Xiu-hong; SHI Xiang-yuan; WU Xian-jun


    Different culture explants, including anther, young panicle, young embryo, and mature embryo, from 19 rice varieties were used for callus induction and green plantlet differentiation. The culture efficiency differed significantly among the four types of explants, and varied from genotype to genotype. Callus induction frequency presented significantly positive correlation each between anther and young panicle, anther and mature embryo, and young panicle and young embryo. Green plantlet differentiation showed no relationship between different types of explants. In addition, no relationship was found between callus induction frequency and green plantlet differentiation frequency.

  16. 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.

  17. Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells. (United States)

    Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo


    One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

  18. Organoid culture systems for prostate epithelial and cancer tissue

    NARCIS (Netherlands)

    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans


    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material cont

  19. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. (United States)

    Kim, B S; Putnam, A J; Kulik, T J; Mooney, D J


    The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were

  20. Characterization of tissue-simulating polymers for photoacoustic vascular imaging (United States)

    Vogt, William C.; Jia, Congxian; Garra, Brian S.; Pfefer, T. Joshua


    Photoacoustic tomography (PAT) is a maturing imaging technique which combines optical excitation and acoustic detection to enable deep tissue sensing for biomedical applications. Optical absorption provides biochemical specificity and high optical contrast while ultrasonic detection provides high spatial resolution and penetration depth. These characteristics make PAT highly suitable as an approach for vascular imaging. However, standard testing methods are needed in order to characterize and compare the performance of these systems. Tissue-mimicking phantoms are commonly used as standard test samples for imaging system development and evaluation due to their repeatable fabrication and tunable properties. The multi-domain mechanism behind PAT necessitates development of phantoms that accurately mimic both acoustic and optical properties of tissues. While a wide variety of materials have been used in the literature, from gelatin and agar hydrogels to silicone, published data indicates that poly(vinyl chloride) plastisol (PVCP) is a promising candidate material for simulating tissue optical and acoustic properties while also providing superior longevity and stability. Critical acoustic properties of PVCP phantoms, including sound velocity and attenuation, were measured using acoustic transmission measurements at multiple frequencies relevant to typical PAT systems. Optical absorption and scattering coefficients of PVCP gels with and without biologically relevant absorbers and scatterers were measured over wavelengths from 500 to 1100 nm. A custom PAT system was developed to assess image contrast in PVCP phantoms containing fluid channels filled with absorbing dye. PVCP demonstrates strong potential as the basis of high-fidelity polymer phantoms for developing and evaluating PAT systems for vascular imaging applications.

  1. Chromatographic study of marmesin and visnagin occurrence in Ammi visnaga Lam. suspension tissue cultures

    Directory of Open Access Journals (Sweden)

    Jadwiga H. Supniewska


    Full Text Available Chromatographic examination of tissue from suspension cultures of A. visnaga proved their ability to biosynthesis of furanochromone-visnagin and furanocoumarin-marmesin. The occurrence of these two compounds depends on the composition of medium which also influences culture growth and embryogenesis, after subculture for at least l year

  2. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    . As the membranes are used for culturing, the introduction of electrodes on these will allow for the real time measurement of relevant cell/tissue products during culturing. The last part of the thesis is about, i.e. the integration of the membrane based sensors with the fluidic system, in a way compatible...

  3. Alginate/polyoxyethylene and alginate/gelatin hydrogels: preparation, characterization, and application in tissue engineering. (United States)

    Aroguz, Ayse Z; Baysal, Kemal; Adiguzel, Zelal; Baysal, Bahattin M


    Hydrogels are attractive biomaterials for three-dimensional cell culture and tissue engineering applications. The preparation of hydrogels using alginate and gelatin provides cross-linked hydrophilic polymers that can swell but do not dissolve in water. In this work, we first reinforced pure alginate by using polyoxyethylene as a supporting material. In an alginate/PEO sample that contains 20 % polyoxyethylene, we obtained a stable hydrogel for cell culture experiments. We also prepared a stable alginate/gelatin hydrogel by cross-linking a periodate-oxidized alginate with another functional component such as gelatin. The hydrogels were found to have a high fluid uptake. In this work, preparation, characterization, swelling, and surface properties of these scaffold materials were described. Lyophilized scaffolds obtained from hydrogels were used for cell viability experiments, and the results were presented in detail.

  4. Pattern matching and adaptive image segmentation applied to plant reproduction by tissue culture (United States)

    Vazquez Rueda, Martin G.; Hahn, Federico


    This paper shows the results obtained in a system vision applied to plant reproduction by tissue culture using adaptive image segmentation and pattern matching algorithms, this analysis improves the number of tissue obtained and minimize errors, the image features of tissue are considered join to statistical analysis to determine the best match and results. Tests make on potato plants are used to present comparative results with original images processed with adaptive segmentation algorithm and non adaptive algorithms and pattern matching.

  5. Spontaneous aneuploidy and clone formation in adipose tissue stem cells during different periods of culturing. (United States)

    Buyanovskaya, O A; Kuleshov, N P; Nikitina, V A; Voronina, E S; Katosova, L D; Bochkov, N P


    Cytogenetic analysis of 13 mesenchymal stem cell cultures isolated from normal human adipose tissue was carried out at different stages of culturing. The incidence of chromosomes 6, 8, 11, and X aneuploidy and polyploidy was studied by fluorescent in situ hybridization. During the early passages, monosomal cells were more often detected than trisomal ones. A clone with chromosome 6 monosomy was detected in three cultures during late passages.

  6. Microbiome Heterogeneity Characterizing Intestinal Tissue and Inflammatory Bowel Disease Phenotype. (United States)

    Tyler, Andrea D; Kirsch, Richard; Milgrom, Raquel; Stempak, Joanne M; Kabakchiev, Boyko; Silverberg, Mark S


    Inflammatory bowel disease has been associated with differential abundance of numerous organisms when compared to healthy controls (HCs); however, few studies have investigated variability in the microbiome across intestinal locations and how this variability might be related to disease location and phenotype. In this study, we have analyzed the microbiome of a large cohort of individuals recruited at Mount Sinai Hospital in Toronto, Canada. Biopsies were taken from subjects with Crohn's disease, ulcerative colitis, and HC, and also individuals having undergone ileal pouch-anal anastomosis for treatment of ulcerative colitis or familial adenomatous polyposis. Microbial 16S rRNA was sequenced using the Illumina MiSeq platform. We observed a great deal of variability in the microbiome characterizing different sampling locations. Samples from pouch and afferent limb were comparable in microbial composition. When comparing sigmoid and terminal ileum samples, more differences were observed. The greatest number of differentially abundant microbes was observed when comparing either pouch or afferent limb samples to sigmoid or terminal ileum. Despite these differences, we were able to observe modest microbial variability between inflammatory bowel disease phenotypes and HCs, even when controlling for sampling location and additional experimental factors. Most detected associations were observed between HCs and Crohn's disease, with decreases in specific genera in the families Ruminococcaceae and Lachnospiraceae characterizing tissue samples from individuals with Crohn's disease. This study highlights important considerations when analyzing the composition of the microbiome and also provides useful insight into differences in the microbiome characterizing these seemingly related phenotypes.

  7. Early fruit setting from tissue culture-derived mangosteen tree


    Sompong Te-chato; Mongkol Lim


    Vitro-plantlets of mangosteen derived from culturing young leaves were acclimatized in 1993. Small and large polybag seedlings were carefully raised under controlled environmental conditions until 1994 when they were ready to be transferred to the field. During this stage, morphological abnormalities of the seedlings were recorded. After transferring to the field for 5-6 years (1994-1999) at Yi Ngo District, Narathiwat Province and Klong Hoi Khong District, Songkhla Province, morphological ch...

  8. 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.

  9. Window-modulated compounding Nakagami imaging for ultrasound tissue characterization. (United States)

    Tsui, Po-Hsiang; Ma, Hsiang-Yang; Zhou, Zhuhuang; Ho, Ming-Chih; Lee, Yu-Hsin


    Ultrasound Nakagami parametric imaging is a useful tool for tissue characterization. Previous literature has suggested using a square with side lengths corresponding to 3 times the transducer pulse length as the minimum window for constructing the Nakagami image. This criterion does not produce sufficiently smooth images for the Nakagami image to characterize homogeneous tissues. To improve image smoothness, we proposed window-modulated compounding (WMC) Nakagami imaging based on summing and averaging the Nakagami images formed using sliding windows with varying window side lengths from 1 to N times the transducer pulse length in 1 pulse length step. Simulations (the number densities of scatterers: 2-16 scatterers/mm(2)) and experiments on fully developed speckle phantoms (the scatterer diameters: 20-106 μm) were conducted to suggest an appropriate number of frames N and to evaluate the image smoothness and resolution by analyzing the full width at half maximum (FWHM) of the parameter distribution and the widths of the image autocorrelation function (ACF), respectively. In vivo ultrasound measurements on rat livers without and with cirrhosis were performed to validate the practical performance of the WMC Nakagami image in tissue characterization. The simulation results showed that using a range of N from 7 to 10 as the number of frames for image compounding reduces the estimation error to less than 5%. Based on this criterion, the Nakagami parameter obtained from the WMC Nakagami image increased from 0.45 to 0.95 after increasing the number densities of scatterers from 2 to 16 scatterers/mm(2). The FWHM of the parameter distribution (bins=40) was 13.5±1.4 for the Nakagami image and 9.1±1.43 for the WMC Nakagami image, respectively (p-valueNakagami and WMC Nakagami images were 454±5.36 and 458±4.33, respectively (p-value>.05). In the phantom experiments, we also found that the FWHM of the parameter distribution for the WMC Nakagami image was smaller than that


    directed to HeLa cells . The following results were obtained: Of those cultures that were susceptible, three different patterns of dose response were...obtained that were interpreted as different possible manifestations of interference. HeLa cells , which were among the least sensitive to low infection

  11. Application of plant tissue cultures in phytoremediation research: incentives and limitations. (United States)

    Doran, Pauline M


    The aim of this review is to critically assess the benefits and limitations associated with the use of in vitro plant cell and organ cultures as research tools in phytoremediation studies. Plant tissue cultures such as callus, cell suspensions, and hairy roots are applied frequently in phytoremediation research as model plant systems. In vitro cultures offer a range of experimental advantages in studies aimed at examining the intrinsic metabolic capabilities of plant cells and their capacity for toxicity tolerance. The ability to identify the contributions of plant cells to pollutant uptake and detoxification without interference from microorganisms is of particular significance in the search for fundamental knowledge about plants. However, if the ultimate goal of plant tissue culture experiments is the development of practical phytoremediation technology, the limitations inherent in the use of in vitro cultures as a representative of whole plants in the field must be recognized. The bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution are likely to be substantially different in the two systems; this can lead to qualitative as well as quantitative differences in metabolic profiles and tolerance characteristics. Yet, many studies have demonstrated that plant tissue cultures are an extremely valuable tool in phytoremediation research. The results derived from tissue cultures can be used to predict the responses of plants to environmental contaminants, and to improve the design and thus reduce the cost of subsequent conventional whole plant experiments.

  12. Physical characterization of hydroxyapatite porous scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Teixeira, S., E-mail: [INEB - Instituto de Engenharia Biomedica, Divisao de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto (Portugal); Universidade do Porto, Faculdade de Engenharia, Departamento de Engenharia Metalurgica e Materiais, Porto (Portugal); Rodriguez, M.A.; Pena, P.; De Aza, A.H.; De Aza, S. [Instituto de Ceramica y Vidrio, CSIC, 28049-Cantoblanco, Madrid (Spain); Ferraz, M.P. [INEB - Instituto de Engenharia Biomedica, Divisao de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto (Portugal); Faculdade de Ciencias da Saude da Universidade Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto (Portugal); Monteiro, F.J. [INEB - Instituto de Engenharia Biomedica, Divisao de Biomateriais, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto (Portugal); Universidade do Porto, Faculdade de Engenharia, Departamento de Engenharia Metalurgica e Materiais, Porto (Portugal)


    The present study refers to the preparation and characterization of porous hydroxyapatite scaffolds to be used as matrices for bone regeneration or as specific release vehicles. Ceramics are widely used for bone tissue engineering purposes and in this study, hydroxyapatite porous scaffolds were produced using the polymer replication method. Polyurethane sponges were used as templates and impregnated with a ceramic slurry at different ratios, and sintered at 1300 deg. C following a specific thermal cycle. The characteristics of the hydroxyapatite porous scaffolds and respective powder used as starting material, were investigated by using scanning electron microscopy, particle size distribution, X-ray diffraction, Fourier transformed infrared spectroscopy and compressive mechanical testing techniques. It was possible to produce highly porous hydroxyapatite scaffolds presenting micro and macropores and pore interconnectivity.

  13. TCUP: A Novel hAT Transposon Active in Maize Tissue Culture. (United States)

    Smith, Alan M; Hansey, Candice N; Kaeppler, Shawn M


    Transposable elements (TEs) are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced TEs that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP), which is transcribed at high levels in long-term maize black Mexican sweet (BMS) tissue culture and is transcribed in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected novel TCUP insertions in two long-term tissue-cultured cell lines of the genotype Hi-II A × B and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  14. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Zhen-Yu Du

    Full Text Available We have developed an in vitro hepatocyte-adipose tissue explant (ATE co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal adipose tissues. Expressions of inflammation related genes (IL-6, TNF-α, COX-2 were higher in the inguinal than the epididymal ATE. Similarly, expressions of marker genes of macrophage and monocyte (MPEG-1, CD68, F4/80, CD64 were higher in the stromal vascular fraction (SVF isolated from inguinal ATE than that from epididymal ATE. However, expressions of lipolysis related genes (ATGL, HSL, perilipin-1 were higher in the epididymal adipocytes than inguinal adipocytes. Moreover, secretion of IL-6 and PGE(2 was higher from inguinal ATEs than from epididymal ATEs. There was a trend that the total levels of IL-6, TNF-α and PGE(2 in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2. Lipolysis, measured as glycerol release, was similar in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs, particularly macrophages, in inguinal adipose tissue resulting in stronger responses in terms of hepatotoxicity and insulin-resistance.

  15. TCUP: A novel hAT transposon active in maize tissue culture

    Directory of Open Access Journals (Sweden)

    Alan eSmith


    Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  16. Versatile electrochemial sensor for tissue culturing and sample handling

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Kwasny, Dorota; Al Atraktchi, Fatima Al-Zahraa;


    in microfluidic devices for sample preparation. In this work we present the development of the sensor system along with results on characterization by impedance spectroscopy and cyclic voltammetry. Furthermore we present recent results on integration of the sensor as well as amperometric detection of dopamine...

  17. Three-dimensional ultrasonic Nakagami imaging for tissue characterization

    Energy Technology Data Exchange (ETDEWEB)

    Tsui, Po-Hsiang [Department of Medical Imaging and Radiological Sciences, Chang Gung University, Taoyuan, Taiwan (China); Hsu, Cheng-Wei; Chang, Chien-Cheng [Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan (China); Ho, Ming-Chih [Department of Surgery, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, Yung-Sheng [Department of Electrical Engineering, Yuan Ze University, Chung Li, Taiwan (China); Lin, Jen-Jen [Department of Applied Statistics and Information Science, Ming Chuan University, Taoyuan, Taiwan (China); Chu, Chin-Chou, E-mail: [Institute of Applied Mechanics, National Taiwan University, Taipei, Taiwan (China)


    The two-dimensional (2D) Nakagami image complements the ultrasound B-scan image when attempting to visualize the scatterer properties of tissues. The resolution of the Nakagami image is lower than that of the B-scan image, since the former is produced by processing the raw envelope data using a 2D sliding window with side lengths typically corresponding to three times the pulse length of the incident ultrasound. This paper proposes using three-dimensional (3D) Nakagami imaging for improving the resolution of the obtained Nakagami image and providing more complete information of scatterers for a better tissue characterization. The 3D Nakagami image is based on a voxel array composed of the Nakagami parameters constructed using a 3D sliding cube to process the 3D backscattered raw data. Experiments on phantoms with different scatterer concentrations were carried out to determine the optimal size of the sliding cube for a stable estimation of the Nakagami parameter. Tissue measurements on rat livers without and with fibrosis formation were further used to explore the practical feasibility of 3D Nakagami imaging. The results indicated that the side length of the cube used to construct the 3D Nakagami image must be at least two times the pulse length, which improved the resolution for each Nakagami image frame in the 3D Nakagami image. The results further demonstrated that the 3D Nakagami image is better than the conventional 2D Nakagami image for complementing the B-scan in detecting spatial variations in the scatterer concentration and classifying normal and fibrotic livers. This study suggests that 3D Nakagami imaging has the potential to become a new 3D quantitative imaging approach.

  18. Three-dimensional ultrasonic Nakagami imaging for tissue characterization (United States)

    Tsui, Po-Hsiang; Hsu, Cheng-Wei; Ho, Ming-Chih; Chen, Yung-Sheng; Lin, Jen-Jen; Chang, Chien-Cheng; Chu, Chin-Chou


    The two-dimensional (2D) Nakagami image complements the ultrasound B-scan image when attempting to visualize the scatterer properties of tissues. The resolution of the Nakagami image is lower than that of the B-scan image, since the former is produced by processing the raw envelope data using a 2D sliding window with side lengths typically corresponding to three times the pulse length of the incident ultrasound. This paper proposes using three-dimensional (3D) Nakagami imaging for improving the resolution of the obtained Nakagami image and providing more complete information of scatterers for a better tissue characterization. The 3D Nakagami image is based on a voxel array composed of the Nakagami parameters constructed using a 3D sliding cube to process the 3D backscattered raw data. Experiments on phantoms with different scatterer concentrations were carried out to determine the optimal size of the sliding cube for a stable estimation of the Nakagami parameter. Tissue measurements on rat livers without and with fibrosis formation were further used to explore the practical feasibility of 3D Nakagami imaging. The results indicated that the side length of the cube used to construct the 3D Nakagami image must be at least two times the pulse length, which improved the resolution for each Nakagami image frame in the 3D Nakagami image. The results further demonstrated that the 3D Nakagami image is better than the conventional 2D Nakagami image for complementing the B-scan in detecting spatial variations in the scatterer concentration and classifying normal and fibrotic livers. This study suggests that 3D Nakagami imaging has the potential to become a new 3D quantitative imaging approach.

  19. Optical characterization of biological tissues and rare earth nanoparticles (United States)

    Barrera, Frederick John, III

    The ubiquitous use of lasers as both a diagnostic and therapeutic tool for medical applications (e.g. laser surgery, photoacoustic imaging, photodynamic therapy etc.), had rendered the understanding of optical properties of a biological medium critically important. The development of biomedical devices for the purposes of imaging or treatment requires a detailed investigation of these properties. Indeed, diagnostic monitoring of blood in vivo depends on knowledge of the distribution of light due to scattering in a blood medium. In addition, many optical properties of tissues have not been investigated experimentally at many clinically relevant wavelengths. The quantification of the scattering and absorptive behavior of tissue and its interaction with electromagnetic radiation is still at the core of predicting the outcome of a desired clinical effect. Therefore, the first portion of this Dissertation is a thorough characterization of ocular tissues in vitro using reflectance and transmittance spectroscopic techniques and computational models to extract and enlist a systematic study at wavelengths in the visible spectral region. The Kubelka-Munk (KM), Inverse Adding Doubling (IAD), and Inverse Monte Carlo (IMC) methods were used to determine the absorption and scattering coefficients and contrasted. The second portion of this Dissertation is an investigation of the optical and spectroscopic properties of novel rare earth Y2O3 and Nd3+:Y2O 3nanoparticles in a blood medium. Reflectance and transmittance measurements were performed and the absorption and scattering properties for the nanoparticle/blood samples were determined by computational methods and compared. Absorption and emission of Y2O3 and Nd3+:Y 2O3nanoparticle/blood medium revealed their utility as biomarkers.

  20. Extraction and characterization of collagen from different biological tissues (United States)

    Gómez, Karla K.; Del Prado, María L.; Piña, M. Cristina; García de León, M. Carmen


    Because many suitable properties, collagen type I is used in medical and cosmetical applications, for this, the collagen extraction from biological tissues as the first source for obtaining this protein is important. We used skin and tail tendon from bovine, and rat tail tendon to obtain collagen type I. Acetic acid was employed to dissolve the collagen from biological tissues, once obtained was characterized using Sodium Dodecyl Sulfate Polyacrilamide Gel Electrophoresis (SDS-PAGE) technique, DSC and SEM. It was found that indeed the collagen type I was obtained. The thermal analysis showed that the denaturation temperature (Td) was 70 °C for all cases and that the folding of the protein at this temperature is irreversible, involving in all cases two steps: an unfolding of the native protein (N) and an irreversible alteration of the unfolded protein (U) to yield a final state (F) that is unable to fold back to the native state. The protein morphology was studied using SEM, it was found that morphology protein is fibrillar. The results suggested that the obtaining process is very efficient because the collagen concentration obtained was very high.

  1. Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests. (United States)

    Cassells, Alan C


    The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

  2. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes

    DEFF Research Database (Denmark)

    Du, Zhen-Yu; Ma, Tao; Lock, Erik-Jan;


    a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs......We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal...... levels of IL-6, TNF-a and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar...

  3. Single molecule microscopy in 3D cell cultures and tissues. (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias


    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

  4. Electron microscopy, tissue culture,and immunology of ovarian carcinoma. (United States)

    Ioachim, H L; Dorsett, B H; Sabbath, M; Barber, H R


    The ultrastructure of the major histologic types of ovarian carcinoma was investigated as part of a multilateral study of this tumor. The nuclear and nucleolar changes in size, shape, and structure correlated well with the degree of malignancy and tumor grading. Cytoplasmic organelles and intercellular junctions were abundant and fairly well differentiated even in ovarian carcinomas of higher grade and stage. Active processes of synthesis and secretion taking place in most of these tumors were suggested by the presence of a richly granulated endoplasmic reticulum, dilated cisternae, and numerous secretory granules. Seventy-eight different ovarian carcinomas of all histologic types were cultured in vitro for periods of up to 300 days, and their morphology in light and electron microscopy was compared to that of the original tumors. The cultures displayed a consistent pattern of growth which led to the conclusion that ovarian cancer cells in vitro preserve their salient features and are representative of the tumors of origin. Heterologous antisera raised with pooled extracts of various types of ovarian carcinomas reacted specifically in immunodiffusion and immunofluorescence tests only with ovarian carcinomas and not with normal ovaries, benigh ovarian tumors, and nonovarian malignant neoplasms, indicating the presence of a cross-reacting specific antigen for ovarian carcinomas. In other studies, autologous antibodies were isolated from antigen-antibody complexes recovered from peritoneal effusions of patients with ovarian carcinomas. These antibodies displayed a high degree of specificity against ovarian carcinoma cells when tested in immunofluorescence assays.

  5. Oil palm (Elaeis guineensis Jacq. tissue culture ESTs: Identifying genes associated with callogenesis and embryogenesis

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    Ooi Leslie CL


    Full Text Available Abstract Background Oil palm (Elaeis guineensis Jacq. is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. Results A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs. The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. Conclusion This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However

  6. Characterization of Diaphanous-related formin FMNL2 in human tissues

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    Kampf Caroline


    Full Text Available Abstract Background Diaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2 in human tissues. Results An FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration. Conclusion FMNL2 is expressed in multiple human tissues, not only in the central nervous system

  7. Clonal propagation of Leptospermum spp. by tissue culture. (United States)

    Shipton, W A; Jackes, B R


    Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04-1.0 μM) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 μM respectively. In L. petersonii ssp. root initiation and development was favoured by β-naphthoxyacetic acid (1 μM) and in L. brachyandrum indole butyric acid and α-naphthalene acetic acid (1 μM) were almost equally effective.

  8. Micropropagation of six Paulownia genotypes through tissue culture

    Directory of Open Access Journals (Sweden)

    Lydia Shtereva


    Full Text Available We investigated the effect of genotype and culture medium on the in vitro germination and development of plantlets from seeds of 6 different Paulownia genotypes (P. tomentosa, hybrid lines P. tomentosa P. fortunei (Mega, Ganter and Caroline, P. elongata and hybrid line P. elongata P. fortunei. Nodal and shoot tip explants were used for micropropagation of Paulownia genotypes by manipulating plant growth regulators. The highest germination percentage for all genotypes was obtained for seeds inoculated on medium supplemented with 50 mg*L GA3 (MSG2. On Thidiazuron containing media, the explants of hybrid line P. elongata P. fortunei exhibited the highest frequency of axillary shoot proliferation following by P. tomentosa P. fortunei. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several economically valuable Paulownia genotypes.

  9. Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique. (United States)

    Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit


    Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

  10. Dissecting mitosis by RNAi in Drosophila tissue culture cells

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    Maiato Helder


    Full Text Available Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique.

  11. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation. (United States)

    Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James


    We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

  12. A Comparison of Tissue versus Swab Culturing of Infected Diabetic Foot Wounds

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    Ying Huang


    Full Text Available Objective. To compare the efficacy of swabbing versus tissue biopsy for microbiological diagnosis of diabetic foot infection. Methods. This was a prospective trial. Fifty-six patients with diabetic foot infection were divided into the following 3 groups according to the PEDIS grading system: grade 2 (n=10, grade 3 (n=29, and grade 4 (n=17. Two specimens were collected from each wound for microbial culturing after debridement, including a superficial swab and a deep tissue punch biopsy specimen. Results. Swab culturing identified all of the microorganisms isolated from the corresponding deep tissue specimens in 9/10 of grade 2 wounds (90.0%, and this proportion decreased to 12/29 (41.4% and 7/17 (41.2% for grades 3 and 4 wounds, respectively (p=0.02. Moreover, the sensitivity for identifying Gram-negative bacteria, such as E. coli and Citrobacter, by swabbing was low (33.3%. In addition, some Gram-negative bacteria, such as Serratia and Ralstonia pickettii, were isolated from deep tissues but not from swabs. Conclusions. Swab culturing may be reliable for identification of pathogens in diabetic foot wounds classified as grade 2. However, it is advisable to culture deep tissue specimens for wounds of grade ≥3 because swab culturing is associated with a high risk of missing pathogens, especially Gram-negative bacteria.

  13. Mesenchymal stem cells from adipose tissue which have been differentiated into chondrocytes in three-dimensional culture express lubricin. (United States)

    Musumeci, Giuseppe; Lo Furno, Debora; Loreto, Carla; Giuffrida, Rosario; Caggia, Silvia; Leonardi, Rosalia; Cardile, Venera


    The present study focused on the isolation, cultivation and characterization of human mesenchymal stem cells (MSCs) from adipose tissue and on their differentiation into chondrocytes through the NH ChondroDiff medium. The main aim was to investigate some markers of biomechanical quality of cartilage, such as lubricin, and collagen type I and II. Little is known, in fact, about the ability of chondrocytes from human MSCs of adipose tissue to generate lubricin in three-dimensional (3D) culture. Lubricin, a 227.5-kDa mucinous glycoprotein, is known to play an important role in articular joint physiology, and the loss of accumulation of lubricin is thought to play a role in the pathology of osteoarthritis. Adipose tissue is an alternative source for the isolation of multipotent MSCs, which allows them to be obtained by a less invasive method and in larger quantities than from other sources. These cells can be isolated from cosmetic liposuctions in large numbers and easily grown under standard tissue culture conditions. 3D chondrocytes were assessed by histology (hematoxylin and eosin) and histochemistry (Alcian blue and Safranin-O/fast green staining). Collagen type I, II and lubricin expression was determined through immunohistochemistry and Western blot. The results showed that, compared with control cartilage and monolayer chondrocytes showing just collagen type I, chondrocytes from MSCs (CD44-, CD90- and CD105- positive; CD45-, CD14- and CD34-negative) of adipose tissue grown in nodules were able to express lubricin, and collagen type I and II, indicative of hyaline cartilage formation. Based on the function of lubricin in the joint cavity and disease and as a potential therapeutic agent, our results suggest that MSCs from adipose tissue are a promising cell source for tissue engineering of cartilage. Our results suggest that chondrocyte nodules producing lubricin could be a novel biotherapeutic approach for the treatment of cartilage abnormalities.

  14. Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression. (United States)

    Gesta, S; Lolmède, K; Daviaud, D; Berlan, M; Bouloumié, A; Lafontan, M; Valet, P; Saulnier-Blache, J S


    Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.


    The tick-borne encephalitis virus is successfully reproduced in tissue cultures of the human embryo, HeLa cells , monkey and dog kidney tissue, skin...tissue and the HeLa cells . A cytopathogenic effect is registered regularly in the cultures of human skin-muscle and kidney tissued on the 2nd-4th day...the embryonic skin-muscle tissue of the white rat. The virus’s cytopathogenic effect is not developed in cultures of human lung tissue, HeLa cells , monkey

  16. Plants regenerated from tissue culture contain stable epigenome changes in rice. (United States)

    Stroud, Hume; Ding, Bo; Simon, Stacey A; Feng, Suhua; Bellizzi, Maria; Pellegrini, Matteo; Wang, Guo-Liang; Meyers, Blake C; Jacobsen, Steven E


    Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:

  17. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure. (United States)

    Talikka, Marja; Kostadinova, Radina; Xiang, Yang; Mathis, Carole; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Merg, Celine; Geertz, Marcel; Martin, Florian; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia


    Exposure to cigarette smoke (CS) is linked to the development of respiratory diseases, and there is a need to understand the mechanisms whereby CS causes damage. Although animal models have provided valuable insights into smoking-related respiratory tract damage, modern toxicity testing calls for reliable in vitro models as alternatives for animal experimentation. We report on a repeated whole mainstream CS exposure of nasal and bronchial organotypic tissue cultures that mimic the morphological, physiological, and molecular attributes of the human respiratory tract. Despite the similar cellular staining and cytokine secretion in both tissue types, the transcriptomic analyses in the context of biological network models identified similar and diverse biological processes that were impacted by CS-exposed nasal and bronchial cultures. Our results demonstrate that nasal and bronchial tissue cultures are appropriate in vitro models for the assessment of CS-induced adverse effects in the respiratory system and promising alternative to animal experimentation.

  18. Tissue culture system using a PANDA ring resonator and wavelength router for hydroponic plant. (United States)

    Kamoldilok, Surachart; Suwanpayak, Nathaporn; Suttirak, Saisudawan; Yupapin, Preecha P


    A novel system of nanofluidics trapping and delivery, which is known as a tissue culture system is proposed. By using the intense optical pulse(i.e., a soliton pulse) and a system constructed by a liquid core waveguide, the optical vortices (gradient optical fields/wells) can be generated, where the trapping tools in the same way as the optical tweezers in the PANDA ring resonator can be formed. By controlling the suitable parameters, the intense optical vortices can be generated within the PANDA ring resonator, in which the nanofluidics can be trapped and moved (transported) dynamically within the Tissue culture system(a wavelength router), which can be used for tissue culture and delivery in the hydroponic plant system.

  19. Obtaining freshly isolated and cultured mesenchymal stem cells from human adipose tissue. (United States)

    Boquest, Andrew C; Collas, Philippe


    The stromal compartment of adipose tissue harbors mesenchymal stem cells (MSCs) (also called stromal stem cells) that display extensive proliferative capacity and multilineage differentiation potential. Such cells offer a practical avenue of generating patient-matched tissue for use in regenerative medicine. It is relatively easy to isolate these cells from adipose tissue in large enough quantities (tens of millions) to allow for their clinical use in a native, uncultured form. Alternatively, MSCs from adipose tissue can be expanded and differentiated into the desired tissue type in vitro using straightforward cell culture techniques. In this chapter, we outline procedures for isolating large numbers of highly purified MSCs from human adipose tissue in their native, uncultured form and methods for their subsequent expansion and differentiation in vitro.

  20. Spheroid culture as a tool for creating 3D complex tissues. (United States)

    Fennema, Eelco; Rivron, Nicolas; Rouwkema, Jeroen; van Blitterswijk, Clemens; de Boer, Jan


    3D cell culture methods confer a high degree of clinical and biological relevance to in vitro models. This is specifically the case with the spheroid culture, where a small aggregate of cells grows free of foreign materials. In spheroid cultures, cells secrete the extracellular matrix (ECM) in which they reside, and they can interact with cells from their original microenvironment. The value of spheroid cultures is increasing quickly due to novel microfabricated platforms amenable to high-throughput screening (HTS) and advances in cell culture. Here, we review new possibilities that combine the strengths of spheroid culture with new microenvironment fabrication methods that allow for the creation of large numbers of highly reproducible, complex tissues.


    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.

  2. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

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    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M. (Medical Univ. School of Silesia, Katowice (Poland))


    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and (14C)thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil.

  3. Molecular markers for genetic stability of intergeneric hybrids Fragaria x Potentilla derived from tissue culture

    Directory of Open Access Journals (Sweden)

    Anca Nicoleta SUTAN


    Full Text Available The effect of growth regulators, explant source and culture age on genetic stability of plants obtained from tissue culture propagation of ornamental strawberry “Serenata” were examined. Genomic DNAs of in vitro-derived shoots and control plant were extracted and compared by RAPD-PCR analyses. Ten primers (from 48 previously tested were selected and used in RAPD analysis to prove the clonal fidelity (i.e. genetic stability of the tissue culture-derived ornamental strawberry plants. The lack of polymorphisms in micropropagated plants screened through molecular markers was used to suggest genetic fidelity. Identicaly banding patterns of the RAPD profiles obtaining from vitroplants, regenerated via organogenesis or meristems culture, suggested that in the ornamental strawberry, variety “Serenata”, neither explant source, nor callus age or limited number of subcultures, in basal media supplemented with low concentration of growth regulators, were associated with occurence of somaclonal variation.

  4. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves (United States)

    Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki


    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

  5. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues. (United States)

    van der Merwe, Mathilde; Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay


    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

  6. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

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    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))


    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.


    Directory of Open Access Journals (Sweden)

    L. P. Khlebova


    Full Text Available Relationship of different morphogenetic processes in immature embryo cultures from 15 spring bread wheat varieties of different ecological and geographical origin was studied. Embryos (14–16 days post anthesis with 1.3–1.5 mm in size were placed with the sculletum upwards on a solid agar medium containing the inorganic components of Linsmaier & Skoog (LS, 3 % sucrose, 2.0 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D. Induced calli were subcultured after 25–30 days interval in fresh medium supplemented with 0.5 mg l-1 2,4-D and 0.5 mg l-1 kinetin. Embryogenic calli were transferred to LS medium containing 0.2 mg l-1 indole-3-acetic acid (IAA. Varietal polymorphism was revealed in relation to callusogenesis, morphogenesis and plant regeneration. The frequency of callusogenesis made 94.3 % with variation from 76.6 % to 100 % depending on a genotype. An active morphogenic process was revealed in 72 % of the varieties tested. The regeneration level depended on the type of morphogenesis (embryoidogenesis, hemmorhizogenesis and rhizogenesis. On average across all varieties it was not high and made 97.9 %; that is one morphogenic line produced about one plant. Organogenesis in 80.2 % of morphogenic calluses did not reach the development stage of the whole plant and stopped with root production.  Plant regeneration by embryoido- and hemmorhizogenesis occurred in 19.8 % of morphogenic calluses. For the study of theoretical aspects of embryoido- and organogenesis as well as genetic transformation of plants the varieties with high regeneration potential are proposed as model objects (Spektr, Skala, Leones, and Zhnitsa. Positive correlation of embryoido-, hemmorhizogenesis and plant regeneration was revealed (r = 0.777, and it proves that there is a common genetic system responsible for those processes. When factorial trait shifted by 1 %, the resultant trait (regeneration increases by 3.59 %. Negative correlation was found between rhizogenesis and

  8. A Comparison of Tissue versus Swab Culturing of Infected Diabetic Foot Wounds


    Ying Huang; Ying Cao; Mengchen Zou; Xiangrong Luo; Ya Jiang; Yaoming Xue; Fang Gao


    Objective. To compare the efficacy of swabbing versus tissue biopsy for microbiological diagnosis of diabetic foot infection. Methods. This was a prospective trial. Fifty-six patients with diabetic foot infection were divided into the following 3 groups according to the PEDIS grading system: grade 2 (n = 10), grade 3 (n = 29), and grade 4 (n = 17). Two specimens were collected from each wound for microbial culturing after debridement, including a superficial swab and a deep tissue punch biops...

  9. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves


    Md. Ziaul Karim; Daisuke Uesugi; Noriyuki Nakayama; M. Monzur Hossain; Kohji Ishihara; Hiroki Hamada


    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for iden...

  10. Dental hard tissue characterization using laser-based ultrasonics (United States)

    Blodgett, David W.; Massey, Ward L.


    Dental health care and research workers require a means of imaging the structures within teeth in vivo. One critical need is the detection of tooth decay in its early stages. If decay can be detected early enough, the process can be monitored and interventional procedures, such as fluoride washes and controlled diet, can be initiated to help re-mineralize the tooth. Currently employed x-ray imaging is limited in its ability to visualize interfaces and incapable of detecting decay at a stage early enough to avoid invasive cavity preparation followed by a restoration. To this end, non-destructive and non-contact in vitro measurements on extracted human molars using laser-based ultrasonics are presented. Broadband ultrasonic waves are excited in the extracted sections by using a pulsed carbon-dioxide (CO2) laser operating in a region of high optical absorption in the dental hard tissues. Optical interferometric detection of the ultrasonic wave surface displacements in accomplished with a path-stabilized Michelson-type interferometer. Results for bulk and surface in-vitro characterization of caries are presented on extracted molars with pre-existing caries.

  11. Phytosteroids from tissue culture of Allium cepa L. and Trachyspermum ammi S prague.

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    Pratibha Chaturvedi


    Full Text Available Production of secondary metabolites by cultured cells provides a particularly important benefit to manipulate and improve the production of desired compounds; thus biotechnological approaches to increase the concentrations of the metabolites are discussed. Present study deals with the production, isolation and identification of phytosterols from tissue culture of Allium cepa and from plant parts and tissue culture of Trachyspermum ammi. Steroidal analysis of plant parts showed the maximum amount of stigmasterol (0.240 mg/gdw which was comparatively little less than that of the amount of β- sitosterol (0.295 mg/gdw in the seeds of T. ammi . The maximum amount of stigmasterol was present in four weeks old tissue of T. ammi (0.249 mg/gdw whereas the highest content of β- sitosterol was observed in six weeks old tissue of A. cepa (0.315 mg/gdw However, lanosterol, was present only in the tissue of A. cepa which was maximum in six weeks old tissue (0.039 mg/gdw.

  12. Detection of genotoxicity in the marine environment: A preliminary feasibility study using primary mussel tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Cornet, Michel [UMR 5805 EPOC ' Environnements et Paleoenvironnements Oceaniques' , Universite Bordeaux 1, CNRS, Avenue des Facultes, 33405 Talence Cedex (France)]. E-mail:


    The purpose of this study was to evaluate the feasibility and potential usefulness of primary cultures of somatic tissues from adult mussel by means of sister chromatid exchange induction (SCE). This research is an initial pilot study carried out with mussel mantle tissue using seawater artificially contaminated with cadmium and polluted seawater from the port of Arcachon. With regard to cadmium concentration, mean SCE numbers showed a progressive increase from 1.07 {+-} 0.18 per diploid cell in controls (i.e. cultures without contaminant) to 2.91 {+-} 0.42 per diploid cell for the highest concentration, 10{sup -4} M. With regard to the medium prepared with seawater from the port of Arcachon, the mean SCE number reached a value of 5.85 {+-} 0.85 per diploid cell. The analysis of SCEs induced by cadmium showed DNA responses even at the lowest concentration (i.e. 10{sup -7} M). The study demonstrates the feasibility of the sister chromatid exchange (SCE) approach based upon primary mussel tissue culture, for the genotoxicity testing of contaminated seawater. Highlights from this procedure are (1) the presence of an active cell proliferation, (2) the use whole-water samples, (3) the possibility of culturing without serum, (4) the absence of cell dissociation before culturing and (5) a cellular proliferation which can be obtained in cultures carried out in a medium containing seawater whose salinity is comprise between 28 and 35 per mille.

  13. Molecular characterization of some lignicolous species from fungal culture collection

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    Stević Nevena


    Full Text Available Culture collections of microorganisms, including fungi, are strain deposits recognised as Biological Resource Centers (BRCs with a great importance in science, industry and education. Their objective is to preserve the purity, viability and genomic integrity of every single strain as a member of such collection. Since improvement of molecular methods nowadays brought many novel approaches in manipulation with strains of microorganisms, they can also be useful for characterization of existing stored strains. ITS1 region in nuclear DNA is preferred barcoding marker for taxon identification, which can be explained by its great inter-species variability. This paper presents results from analysing ITS1 region sequences (17 obtained from fungal DNA of culture collection of autochthonous, lignicolous genera Piptoporus, Pleurotus, Ganoderma and Schizophyllum cultured on malt agar plates for 14 days at 25°C. BLAST (Basic Local Alignment Search Tool was used for comparison with online databases, while alignment of sequences was made with MEGA 5.10 software. Morphological determination of species or genus was confirmed for 13 cultures, while the others were disproved. The resulting alignment indicated small intra-species variability of ITS1 region and pointed to it as an ideal marker for verification of fungal culture collections' authenticity. [Projekat Ministarstva nauke Republike Srbije, br. III43002 and by the Provincial Secretariat for Science and Technological Development, Vojvodina, Serbia APV 114-4513592/2013-03: Molecular and phenotypic diversity of taxa of economical and epidemiological importance, and endangered and endemic species in Europe


    Directory of Open Access Journals (Sweden)

    Lila Gardenia


    Full Text Available Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin. Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Primary cells infected with koi herpes virus produced typical cytopathic effects characterized by severe vacuolation and deformation of nuclei, which is consistent with those of previous reports. Artificial injection experiment by using supernatant koi herpes virus SKBM-1 isolate revealed that it could cause 90% mortality in infected fish within two weeks. PCR test with Sph I-5 specific primers carried out with DNA template from supernatant virus, pellet cell, and gills of infected fish showed positive results in all samples (molecular weight of DNA target 290 bp. The cells were found to be susceptible to koi herpes virus and can be used for virus propagation.

  15. Phytoceramide in vertebrate tissues: one step chromatography separation for molecular characterization of ceramide species.

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    Somsankar Dasgupta

    Full Text Available Ceramide is a precursor for complex sphingolipids in vertebrates, while plants contain phytoceramide. By using a novel chromatography purification method we show that phytoceramide comprises a significant proportion of animal sphingolipids. Total ceramide including phytoceramide from mouse tissue (brain, heart, liver lipid extracts and cell culture (mouse primary astrocytes, human oligodendroglioma cells was eluted as a single homogenous fraction, and then analyzed by thin layer chromatography, and further characterized by gas chromatography-mass spectrometry (GC-MS. We detected a unique band that migrated between non-hydroxy fatty acyl ceramide and hydroxy fatty acyl ceramide, and identified it as phytoceramide. Using RT-PCR, we confirmed that mouse tissues expressed desaturase 2, an enzyme that has been reported to generate phytoceramide from dihydroceramide. Previously, only trace amounts of phytoceramide were reported in vertebrate intestine, kidney, and skin. While its function is still elusive, this is the first report of phytoceramide characterization in glial cells and vertebrate brain, heart, and liver.

  16. In situ fiber-optical monitoring of cytosolic calcium in tissue explant cultures

    CERN Document Server

    Ryser, Manuel; Geiser, Marianne; Frenz, Martin; Rička, Jaro


    We present a fluorescence-lifetime based method for monitoring cell and tissue activity in situ, during cell culturing and in the presence of a strong autofluorescence background. The miniature fiber-optic probes are easily incorporated in the tight space of a cell culture chamber or in an endoscope. As a first application we monitored the cytosolic calcium levels in porcine tracheal explant cultures using the Calcium Green-5N (CG5N) indicator. Despite the simplicity of the optical setup we are able to detect changes of calcium concentration as small as 2.5 nM, with a monitoring time resolution of less than 1 s.

  17. 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.

  18. Biomimetic injectable HUVEC-adipocytes/collagen/alginate microsphere co-cultures for adipose tissue engineering. (United States)

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


    Engineering adipose tissue that has the ability to engraft and establish a vascular supply is a laudable goal that has broad clinical relevance, particularly for tissue reconstruction. In this article, we developed novel microtissues from surface-coated adipocyte/collagen/alginate microspheres and human umbilical vein endothelial cells (HUVECs) co-cultures that resembled the components and structure of natural adipose tissue. Firstly, collagen/alginate hydrogel microspheres embedded with viable adipocytes were obtained to mimic fat lobules. Secondly, collagen fibrils were allowed to self-assemble on the surface of the microspheres to mimic collagen fibrils surrounding the fat lobules in the natural adipose tissue and facilitate HUVEC attachment and co-cultures formation. Thirdly, the channels formed by the gap among the microspheres served as the room for in vitro prevascularization and in vivo blood vessel development. The endothelial cell layer outside the microspheres was a starting point of rapid vascular ingrowth. Adipose tissue formation was analyzed for 12 weeks at 4-week intervals by subcutaneous injection into the head of node mice. The vasculature in the regenerated tissue showed functional anastomosis with host blood vessels. Long-term stability of volume and weight of the injection was observed, indicating that the vasculature formed within the constructs benefited the formation, maturity, and maintenance of adipose tissue. This study provides a microsurgical method for adipose regeneration and construction of biomimetic model for drug screening studies.

  19. 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.

  20. Soil water requirements of tissue-cultured Dwarf Cavendish banana ( Musa spp. L) (United States)

    Shongwe, V. D.; Tumber, R.; Masarirambi, M. T.; Mutukumira, A. N.

    The banana is one of the most important fruit crops in the world. In terms of consumption, the banana fruit is ranked high yet there has not been much research particularly in relation to water requirements for propagules produced by tissue culture. In recent years, tissue culture banana planting material has become increasingly important due to its vigorous growth and high yields. The objective of this study was to investigate optimum soil water requirements of tissue-cultured banana. Dwarf Cavendish tissue-cultured plantlets grown in pots in a greenhouse were subjected to four irrigation regimes at 100% ETm, 85% ETm, 65% ETm, and 40% ETm. Plant parameters measured were leaf number, plant height, pseudo-stem girth, leaf length, leaf width, leaf area, leaf area index, leaf index, leaf colour, and plant vigour. Soil water potential measurements were also made over a three-month period. Differences between irrigating at 100% ETm and 85% ETm were not significantly ( P plant height, and plant height, compared to 65% and 40% ETm treatments. Pseudo-stem girth was highest from the 100% ETm compared to the other treatments. Economic yields of banana may be obtained with irrigation regimes ranging between 100% ETm and 85% ETm.

  1. Amending Storage Vessel and Media Improves Subculture Interval of Musa sp. Tissue Culture Plantlets (United States)

    Bananas and plantains (Musa sp.) are some of the most important food crops in the world. The USDA-ARS, Tropical Agriculture Research Station Musa spp. collection consists of 140 accessions maintained as clonally propagated plants in field plots as well as in tissue culture. Accessions maintained i...

  2. Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

    NARCIS (Netherlands)

    Smulders, M.J.M.; Rus-Kortekaas, W.; Vosman, B.


    The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that

  3. Extended metAFLP approach in studies of tissue culture induced variation (TCIV) in triticale. (United States)

    Machczyńska, Joanna; Orłowska, Renata; Zimny, Janusz; Bednarek, Piotr Tomasz


    We present the development of the theoretical background of the metAFLP approach which allows for partition of complex variation into sequence changes, de novo methylation and demethylation of the regenerants derived via in vitro tissue culture methods in the case of triticale. It was demonstrated that, independent of whether andro- or embryogenesis was used for plant regeneration, the level of sequence changes identified between regenerants is about 10 %. Moreover, DNA demethylation prevails over de novo methylation of the regenerants compared to the donor plant. The metAFLP approach allows for the evaluation of numerous quantitative characteristics. For instance, one may quantify the number of sites unaffected by tissue culture approaches, global site DNA methylation etc. It is suggested that the approach could be useful for breeders in order to control plant material uniformity or for the evaluation of modified in vitro tissue culture approaches allowing for control of the (epi)mutation level. The extended metAFLP approach presented here delivers sufficient background for the evaluation of software that could facilitate analyses of the tissue culture induced variation.

  4. The chemical constituents of the tissue culture cells of Daphne giraldii cullus

    Institute of Scientific and Technical Information of China (English)

    Zhao Hua Wu; Li Bo Wang; Hui Yuan Gao; Jian Huang; Bo Hang Sun; Shu Hui Li; Li Jun Wu


    Three compounds were isolated from the tissue culture cells of Daphne giraldii cullus, their structures were identified as daphneolone (1), S-(+)-1-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-5-phenyl-1-pentanone (2), S-(+)-1-(4-methoxyphenyl)-3-hydroxy-5-phenyl-1-pentanone (3), and among them, 2 was a new compound, 3 was a novel natural product.

  5. Implementing oxygen control in chip-based cell and tissue culture systems

    NARCIS (Netherlands)

    Oomen, Pieter; Skolimowski, Maciej; Verpoorte, Elisabeth


    Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-ch

  6. in tissue culture of lilium explants may become heavily contaminated by the standard initiation procedure

    NARCIS (Netherlands)

    Askari Rabori, N.; Wang, Y.G.; Klerk, de G.J.M.


    In tissue culture of Lilium, the standard initiation procedure brought about substantial contamination in two ways. (1) When scales were detached from the mother bulb, microorganisms could enter via the wound. This source of contamination was strongly enhanced by the negative hydrostatic pressure wi

  7. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells. (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B


    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  8. Thermal compression and characterization of three-dimensional nonwoven PET matrices as tissue engineering scaffolds. (United States)

    Li, Y; Ma, T; Yang, S T; Kniss, D A


    Nonwoven fibrous matrices have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The method of thermal compression of nonwoven polyethylene terephthalate (PET) fabrics was developed and key parameters of temperature, pressure, and compression duration were evaluated in this study. The permanent deformation was obtained at elevated temperature under pressure and the viscoelastic compressional behaviors were observed, characterized by a distinct apparent modulus change in glass transition temperature region. A liquid extrusion method was further employed to analyze both pore size and its distribution for matrices with porosity ranging from 84 to 93%. It is also found that a more uniformly distributed pore size was resulted from thermal compression and the isotropic nature of nonwoven fabrics was preserved because of the proportional reduction of the pore by compression. The thermally compressed fabric matrices with two different pore sizes (15 and 20 microm in pore radius) were used to culture human trophoblast ED27 and NIH 3T3 cells. It was found that cells cultured in the different pore-size PET matrices had different cell spatial organization and proliferation rates. The smaller pores in the matrix allowed cells to spread better and proliferate faster, while cells in the larger pores tended to form large aggregates and had lower proliferation rate. The thermal compression technique also can be applied to other synthetic fibrous matrices including biodegradable polymers used in tissue engineering to modify the microstructure according to their viscoelastic properties.

  9. Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue. (United States)

    Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro


    Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies

  10. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues. (United States)

    Toyoda, Mito; Matsubara, Yoshinori; Lin, Konghua; Sugimachi, Keizou; Furue, Masutaka


    Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue-derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31(-)CD34(+)CD45(-)CD90(-)CD105(-)CD146(+) population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31(-)CD34(+)CD45(-)CD90(-)CD105(-)CD146(-) population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood-derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose-derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue.

  11. Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane. (United States)

    Vilela, J M V; Leonel, E C R; D'Oliveira, L; Paiva, R E G; Miranda-Vilela, A L; Amorim, C A; Pic-Taylor, A; Lucci, C M


    In vitro culture and transplantation procedures are essential protocols employed in the evaluation of ovarian follicle survival and development. Culture in the chorioallantoic membrane (CAM) of chick embryos is an intermediate method that provides important follicle development information and has not been tested for cat ovaries to date. The aim of this study was to investigate if in vitro and CAM culture could be used as short-term systems to study cat ovarian tissue development. The ovaries of eight cats were dissected into 3-mm(3) cubes, cultured in vitro and in CAM for up to 5 days, and stained with hematoxylin-eosin and Gomori trichrome. Cell proliferation was analyzed using anti-Ki67. Possible differences among groups were investigated by analysis of variance or the Kruskal-Wallis test followed by Bonferroni correction. The T-test or Wilcoxon test was used to verify differences between the CAM and IVC. Results revealed that 87.5% of all follicles were primordial during culture. The percentage of primordial follicles in the morphologically normal follicles (MNF) pool was always higher than 80%, with the exception of Day 3 of CAM culture, but the number of MNF reduced significantly from Day 0 (600 out of 777 follicles) to Day 5 in the CAM (91 out of 171) and IVC (296 out of 686). The number of primordial follicles in 1 mm(3) in Days 2, 3, and 5 in the CAM was significantly lower than that in the control (Day 0). No cellular proliferation was observed in culture. Vascularization occurred in the CAM culture, but with no association to follicular viability. In addition, both methods showed an increase in connective tissue during culture. Although no significant differences were observed in the percentage of MNF, there was a reduction in the total number of follicles, both for IVC and CAM-cultured ovarian tissue. Furthermore, anti-Ki67 did not stain any follicle after Day 0 in IVC or in CAM culture. Neither system was capable of promoting follicle growth and

  12. Optimizing culture medium for meristem tissue culture of several Saccharum species and commercial hybrids (United States)

    The optimal range of medium nutrients and plant growth regulators (PGR) was investigated for in vitro culture of diverse sugarcane species and cultivars. Macro-nutrients, nitrogen (N), phosphorous (P) and potassium (K), were essential for growth of leaf primordia. Although the best concentration of ...

  13. Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.

    Directory of Open Access Journals (Sweden)

    Bednarek Piotr T


    Full Text Available Abstract Background When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level. Results A variant of methylation sensitive AFLP, based on the isoschizomeric combinations Acc65I/MseI and KpnI/MseI was applied to analyze, at both the sequence and methylation levels, the outcomes of regeneration from tissue culture in barley. Both sequence mutation and alteration in methylation pattern were detected. Two sets of regenerants from each of five DH donor lines were compared. One set was derived via androgenesis, and the other via somatic embryogenesis, developed from immature embryos. These comparisons delivered a quantitative assessment of the various types of somaclonal variation induced. The average level of variation was 6%, of which almost 1.7% could be accounted for by nucleotide mutation, and the remainder by changes in methylation state. The nucleotide mutation rates and the rate of epimutations were substantially similar between the andro- and embryo-derived sets of regenerants across all the donors. Conclusion We have developed an AFLP based approach that is capable of describing the qualitative and quantitative characteristics of the tissue culture-induced variation. We believe that this approach will find particular value in the study of patterns of inheritance of somaclonal variation, since non-heritable variation is of little interest for the improvement of plant

  14. Myocardial ultrasonic tissue characterization in patients with thyroid dysfunction

    Directory of Open Access Journals (Sweden)

    Schmidt André


    Full Text Available Abstract Background Structural myocardial abnormalities have been extensively documented in hypothyroidism. Experimental studies in animal models have also shown involvement of thyroid hormones in gene expression of myocardial collagen. This study was planned to investigate the ability of ultrasonic tissue characterization, as evaluated by integrated backscatter (IBS, to early identify myocardial involvement in thyroid dysfunction. Patients and Methods We studied 15 patients with hyperthyroidism (HYPER, 8 patients with hypothyroidism (HYPO, 14 patients with subclinical hypothyroidism (SCH and 19 normal (N subjects, who had normal LV systolic function. After treatment, 10 HYPER, 6 HYPO, and 8 SCH patients were reevaluated. IBS images were obtained and analyzed in parasternal short axis (papillary muscle level view, at left ventricular (LV posterior wall. The following IBS variables were analyzed: 1 the corrected coefficient (CC of IBS, obtained by dividing IBS intensity by IBS intensity measured in a rubber phantom, using the same equipment adjustments, at the same depth; 2 cardiac cyclic variation (CV of IBS - peak-to-peak difference between maximal and minimal values of IBS during cardiac cycle; 3 cardiac cyclic variation index (CVI of IBS - percentual relationship between the cyclic variation (CV and the mean value of IBS intensity. Results CC of IBS was significantly larger (p Conclusions CC of IBS was able to differentiate cardiac involvement in patients with overt HYPO and HYPER who had normal LV systolic function. These early myocardial structural abnormalities were partially reversed by drug therapy in HYPER group. On the other hand, although mean IBS intensity tended to be slightly larger in patients with SCH as compared to N, this difference was not statistical significant.

  15. Effect of adipose tissue processing procedures in culture result: a study preliminary

    Directory of Open Access Journals (Sweden)

    Jeanne A. Pawitan


    Full Text Available Background: There are various methods of processing adipose tissue before culture, depending on the adipose tissue samples. The aim of this study is to compare several modifications of culturing and sub-culturing procedures of adipose tissue to fit the condition in our laboratory.Method: This is a descriptive study that was done in the Immunology and Endocrinology Integrated Laboratory, University of Indonesia, from  October 2009 to April 2010. Three adipose tissue processing procedures, various amount of seeding and two subculture methods were compared in term of cell yield and time needed. In the first procedure, collagenase-1 digestion was done in 30minutes, cell seeding were 24,000 and 36,000 per flask; in the second procedure, collagenase-1 digestion was done in 60minutes, cell seeding were 24,000, 48,000, and 72,000 per flask; and in the third procedure, the adipose tissue remnants from the first  procedure were again digested for another 45 minutes, cell seeding were 74,000, and 148,000 per flask. Difference in subculture methods were the presence or absence of washing step.Result: Procedure 1 yielded the lowest amount of cell, and after culture, the cells grew very slow, and was contaminated before harvest of primary culture. Procedure-2 and -3 succeeded to yield primary cultures. Some of the cultures were contaminated, so that further subculture was not  applicable, and only one tissue processing procedure (procedure 2: 60 minute collagenase-1 digestion, without lysis buffer, cell seeding 48,000 and 72,000 could complete the three subcultures. Though some of the procedures could not be completed, final result could be concluded.Conclusion: In this preliminary study, 60 minute colagenase-1 digestion with intermittent shaking every 5 minutes and cell seeding around 50,000 or more, followed by subculture method without washing step gave the best result. (Med J Indones 2011; 20:15-9Keywords: collagenase-1, primary culture, subculture

  16. Hyperspectral Image Analysis Algorithm for Characterizing Human Tissue


    Wondim, Yonas kassaw


    AbstractIn the field of Biomedical Optics measurement of tissue optical properties, like absorption, scattering, and reduced scattering coefficient, has gained importance for therapeutic and diagnostic applications. Accuracy in determining the optical properties is of vital importance to quantitatively determine chromophores in tissue.There are different techniques used to quantify tissue chromophores. Reflectance spectroscopy is one of the most common methods to rapidly and accurately charac...

  17. 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.

  18. Feasibility and accuracy of tissue characterization with dual source computed tomography

    NARCIS (Netherlands)

    Van Abbema, Joanne K.; Van der Schaaf, Arjen; Kristanto, Wisnu; Groen, Jaap M.; Greuter, Marcel J. W.


    PURPOSE: To evaluate the feasibility and accuracy of a model for tissue characterization with dual source computed tomography (DSCT). METHODS AND MATERIALS: A model for tissue characterization in CT was used with a parameterization of linear attenuation coefficients. Sixteen chemical substances with

  19. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues. (United States)

    de Bekker, Charissa; Smith, Philip B; Patterson, Andrew D; Hughes, David P


    Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  20. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    Directory of Open Access Journals (Sweden)

    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  1. Resistin induces lipolysis and suppresses adiponectin secretion in cultured human visceral adipose tissue. (United States)

    Chen, Neng; Zhou, Lingmei; Zhang, Zixiang; Xu, Jiaying; Wan, Zhongxiao; Qin, Liqiang


    Resistin is an adipokine secreted from adipose tissue, which is likely involved in the development of obesity and insulin resistance via its interaction with other organs, as well as affecting adipose tissue function. The impact of resistin treatment on lipolysis and adiponectin secretion in human visceral adipose tissue is currently unknown. Mesenteric adipose tissue samples were obtained from 14 male subjects [age 54±6 yr, body mass index (BMI) 23.59±0.44 kg/m(2)] undergoing abdominal surgeries. Adipose tissues were cultured and treated with resistin (100 ng/mL, 24h) in the absence or presence of different signaling inhibitors: H89 (1 μM), PD98059 (25 μM) and SB201290 (20 μM) for glycerol and non-esterified fatty acid (NEFA) measurement. Adiponectin level from media at 24 h was also measured via ELISA. Adipose tissue minces after resistin incubation (100 ng/mL, 24 h) were also collected for further Western blotting analysis. Resistin resulted in significant induction of glycerol (3.62±0.57 vs. 5.30±1.11 mmol/L/g tissue, ptissue, ptissue, ptissues via its effect on adipose tissue function.

  2. Characterization of the human visceral adipose tissue secretome

    NARCIS (Netherlands)

    Alvarez Llamas, Gloria; Szalowska, Ewa; de Vries, Marcel P.; Weening, Desiree; Landman, Karloes; Hoek, Annemieke; Wolffenbuttel, Bruce H. R.; Roelofsen, Johan; Vonk, Roel J.


    Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and t

  3. Ultrasonic characterization of porcine liver tissue at frequency between 25 to 55 MHz

    Institute of Scientific and Technical Information of China (English)

    Xiao-Zhou Liu; Xiu-Fen Gong; Dong Zhang; Shi-Gong Ye; Bing Rui


    AIM: To study the relation between acoustic parameters and histological structure of biological tissue and to provide the basis for high-resolution image of biological tissues and quantitative ultrasonic diagnosis of liver disease.METHODS: Ultrasonic imaging and tissue characterization of four normal porcine liver and five cirrhotic liver tissue samples were performed using a high frequency imaging system.RESULTS: The acoustic parameters of cirrhotic liver tissue were larger than those of normal liver tissue. The sound velocity was 1577 m/s in normal liver tissue and 1631 m/s in cirrhotic liver tissue. At 35 MHz, the attenuation coefficient was 3.0 dB/mm in normal liver tissue and 4.1 dB/mm in cirrhotic liver tissue. The backscatter coefficient was 0.00431 dB/Srmm in cirrhotic liver tissue and 0.00303 dB/Srmm in normal liver tissue. The backscatter coefficient increased with the frequency. The high frequency images coincided with their histological features.CONCLUSION: The acoustic parameters, especially the sound backscatter coefficient, are sensitive to the changes of liver tissues and can be used to differentiate between the normal and pathological liver tissues.High frequency image system is a useful device for high-resolution image and tissue characterization.

  4. Effects of air pollutants on plant cell tissue cultures. [Tobacco, rose soybean, periwinkle, and morning glory

    Energy Technology Data Exchange (ETDEWEB)


    Experiments were conducted to determine morphological and physiological effects of air pollutants on plant tissue cultures. Several cultures will be exposed to polluted atmospheres for various periods and observed for effects. The cultures which have been developed for this purpose are: tobacco pith, rose stem, soybean stem, periwinkle, and morning glory. Exposures will follow two regimens: a relatively high concentration of pollutant for a short duration and a low concentration for a long duration. Effects of pollutants on cell morphology will be observed microscopically. Effects on cell physiology may include altered respiratory quotients which will be determined by Warburg respirometry techniques. The design of an apparatus that is being developed to mix a pollutant with air and deliver it to the cultures is described.

  5. Studies on the reaction in tissue culture of tomato genotypes under biotic stress

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    Ewa Hanus-Fajerska


    Full Text Available Plant regeneration in vitro from virus-infected somatic tomato (Lycopersicon sp. tissue was performed. Regeneration experiments were started after the determination of virus presence, using enzyme-linked immunosorbent assay, in leaves used as a source of explants. Leaf explants infected with selected strains of tomato mosaic Tobamovirus or cucumber mosaic Cucumovirus respectively, were cultured on a standarised MS agar medium to induce adventitious shoots, which were afterwards excised, rooted in vitro and cultured to plants. Explants were also screened for their ability to produce callus. Diverse effects of viral infection, ranging from stimulation to inhibition of callus formation and of morphogenesis rate, were observed. The health condition of the tissue proved to affect regeneration potential of Lycopersicon esculentum, whereas wild accesions did not react in that case so distinctly. In cultivated tomato was encountered the decline in competence to reproduce shoots adventitiously in infected tissue. There was also relationship between donor plant health condition and adventitious root formation in regenerated shoots. Experiments with short-term cultures of L. esculenum reveled also that a certain number of shoots regenerated from diseased tissue can be virus-free.

  6. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

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    Lindolfo da Silva Meirelles


    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747.

  7. Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata. (United States)

    O'Neill, Matthew; Gaume, Béatrice; Denis, Françoise; Auzoux-Bordenave, Stéphanie


    Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO3) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.

  8. Organ and tissue donation in migrants: advanced course for cross-cultural mediators. (United States)

    Potenza, R; Guermani, A; Grosso, M; Fossarello, L; Fontaneto, C; Casciola, A; Donadio, P P


    Between 2004 and 2010 in Piedmont (Italy Northern Region) 1556 brain-death situations were reported, including 113 (7.3%) in migrants as potential organ and tissue donors. The health staff often has to face migrants, who show great cultural differences and language difficulties. The Molinette Hospital Customer Care Service, the Piedmont Regional Tissue and Organ Procurement Coordination Agency (RPC), and the Cross-Cultural Mediators Association (CMA) organized a special course for intercultural mediators, to decrease misunderstandings between the health staff and the migrants' families and to improve professional communication. In 2011, 28 cultural-linguistic mediators representing different groups of migrants in Piemonte took part in a specific course. Over a 5 month period they were informed about emotional and communicative aspects, proper to the moment of death, as well as organ donation as an intercultural field, the professional role of the mediator, the clinical and forensic aspects of brain death and donation, and the psychological aspects of organ donation. The course was organized by cultural-linguistic mediators of the CMA, the staff of the RPC and the teachers at Turin University. The list of the 21 mediators who passed the final exam was given to organ and tissue donation hospital co-ordinators in Piedmont, so that if necessary, they could obtain the cooperation of these qualified people.

  9. A Protocol for Rapid, Measurable Plant Tissue Culture Using Stem Disc Meristem Micropropagation of Garlic ("Allium Sativum L.") (United States)

    Peat, Gerry; Jones, Meriel


    Plant tissue culture is becoming an important technique for the mass propagation of plants. Problems with existing techniques, such as slow growth and contamination, have restricted the practical work in plant tissue culture carried out in schools. The new protocol using garlic meristematic stem discs explained in this article addresses many of…

  10. Characterization of electrical stimulation electrodes for cardiac tissue engineering. (United States)

    Tandon, Nina; Cannizzaro, Chris; Figallo, Elisa; Voldman, Joel; Vunjak-Novakovic, Gordana


    Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. The goal of this study was to assess the conditions of electrical stimulation with respect to the electrode geometry, material properties and charge-transfer characteristics at the electrode-electrolyte interface. We compared various biocompatible materials, including nanoporous carbon, stainless steel, titanium and titanium nitride, for use in cardiac tissue engineering bioreactors. The faradaic and non-faradaic charge transfer mechanisms were assessed by electrochemical impedance spectroscopy (EIS), studying current injection characteristics, and examining surface properties of electrodes with scanning electron microscopy. Carbon electrodes were found to have the best current injection characteristics. However, these electrodes require careful handling because of their limited mechanical strength. The efficacy of various electrodes for use in 2-D and 3-D cardiac tissue engineering systems with neonatal rat cardiomyocytes is being determined by assessing cell viability, amplitude of contractions, excitation thresholds, maximum capture rate, and tissue morphology.

  11. An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds

    Directory of Open Access Journals (Sweden)

    Alexander Röder


    Full Text Available The use of biopolymers as a three dimensional (3D support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface properties to assess whether seeding efficiencies and cell growth on 3D scaffolds were affected. Cell attachment and growth of murine calvarial osteoblast (MC3T3-E1 cells within a melt-electrospun poly-ε-caprolactone scaffold were assessed when cultured in either “low-adhesive” non-treated or corona discharged-treated well-plates. Increased cell adhesion was observed on the scaffold placed in the surface treated culture plates compared to the scaffold in the non-treated plates 24 h after seeding, although it was not significant. However, higher cell metabolic activity was observed on the bases of all well-plates than on the scaffold, except for day 21, well metabolic activity was higher in the scaffold contained in non-treated plate than the base. These results indicate that there is no advantage in using non-treated plates to improve initial cell seeding in 3D polymeric tissue engineering scaffolds, however non-treated plates may provide an improved metabolic environment for long-term studies.

  12. Plant tissue culture--an opportunity for the production of nutraceuticals. (United States)

    Lucchesini, Mariella; Mensuali-Sodi, Anna


    This chapter provides a short discussion about the opportunity to cultivate in vitro plant tissue of species which synthesize secondary metabolites of nutraceutical interest. The introduction of species of particular interest in cultivation and domestication, can be an alternative to the harvest of wild species. In vitro culture techniques are a useful tool to improve production and marketing nutraceutical species which allows to make a rapid clonal propagation of plants selected for their active principles. The techniques of tissue culture are described in detail. In particular, it is underlined the necessity to clone selected plants and produce true-type plants when standardized plant products are the main goal. This can be reached by conventional micropropagation protocols culturing plants in vitro through the five culture phases. Another approach consists in applying unconventional systems in the last phase of in vitro culture which permit to develop autotrophy of the explants. Autotrophic growth improves the quality of the multiplied shoots and facilitates the acclimatization of the plantlets.

  13. Electrical impedance characterization of normal and cancerous human hepatic tissue. (United States)

    Laufer, Shlomi; Ivorra, Antoni; Reuter, Victor E; Rubinsky, Boris; Solomon, Stephen B


    The four-electrode method was used to measure the ex vivo complex electrical impedance of tissues from 14 hepatic tumors and the surrounding normal liver from six patients. Measurements were done in the frequency range 1-400 kHz. It was found that the conductivity of the tumor tissue was much higher than that of the normal liver tissue in this frequency range (from 0.14 +/- 0.06 S m(-1) versus 0.03 +/- 0.01 S m(-1) at 1 kHz to 0.25 +/- 0.06 S m(-1) versus 0.15 +/- 0.03 S m(-1) at 400 kHz). The Cole-Cole models were estimated from the experimental data and the four parameters (rho(0), rho(infinity), alpha, f(c)) were obtained using a least-squares fit algorithm. The Cole-Cole parameters for the cancerous and normal liver are 9 +/- 4 Omega m(-1), 2.2 +/- 0.7 Omega m(-1), 0.5 +/- 0.2, 140 +/- 103 kHz and 50 +/- 28 Omega m(-1), 3.2 +/- 0.6 Omega m(-1), 0.64 +/- 0.04, 10 +/- 7 kHz, respectively. These data can contribute to developing bioelectric applications for tissue diagnostics and in tissue treatment planning with electrical fields such as radiofrequency tissue ablation, electrochemotherapy and gene therapy with reversible electroporation, nanoscale pulsing and irreversible electroporation.

  14. Cell culture density affects the proliferation activity of human adipose tissue stem cells. (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee


    In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions.

  15. Ontogenetically-regulated male sterility in tissue culture - induced and spontaneous sorghum mutants

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    Elkonin L.A.


    Full Text Available Variability of male fertility expression in the AS-1 line, a somaclonal variant obtained from tissue culture of CMS-plant, and in the progeny of revenant '124-1' obtained from fertile tiller, which developed on CMS-plant transferred from the field to the greenhouse, was investigated. Both revertants were characterized by similar expression of male fertility during plant ontogenesis: the panicle on the main tiller was almost completely sterile whereas formation of fertile pollen grains and seed set were observed on the panicles of the shoot tillers. A clear basipetal gradient of male fertility was manifested on all panicles: the base had significantly higher per cent of fertile pollen grains in comparison with the middle part, while in the top the anthers were either absent or had few sterile pollen grains. Such an ontogenetically-regulated restoration of male fertility was controlled by nuclear genes and could be transferred through the pollen in crosses with progenitor CMS-line. Growing of AS-1 plants in the growth chambers simultaneously under a long (16/8 and a short (12/12 daylength conditions demonstrated that differences of fertility level in different tillers was not caused by change of photoperiod during plant ontogenesis and functioning of photoperiod-sensitive fertility restoring gene. Whereas, the ontogenetically-regulated expression of male fertility in both revenants was temperature-dependent and was clearly manifested under relatively cool conditions during 2-week period before the beginning of anthesis of the first panicle (average daily temperature 21°C. The increase of the average daily temperature by 2-3 С resulted in sharp increase of male fertility level. Possibility of using AS-1 line in a new "two-line system" of hybrid seed production, which require only two lines (sterile mutant and fertility restorer, is discussed.

  16. Synthesis and characterization of a novel chitosan/montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Katti, Kalpana S; Katti, Dinesh R; Dash, Rajalaxmi [Department of Civil Engineering, North Dakota State University, Fargo, ND 58105 (United States)], E-mail:


    Recently, biopolymer-based nanocomposites have been replacing synthetic polymer composites for various biomedical applications. This is often because of the biocompatible and biodegradable behavior of natural polymers. Several studies have been reported pertaining to the synthesis and characterization of chitosan(chi)/montmorillonite(MMT) and chitosan (chi)/hydroxyapatite (HAP) for tissue engineering applications. In the present work, a biopolymer-based novel nanocomposite chitosan/montmorillonite (MMT)/hydroxyapatite (HAP) was developed for biomedical applications. The composite was prepared from chitosan, unmodified MMT and HAP precipitate in aqueous media. The properties of the composites were investigated using x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and thermogravimetric analysis (TGA). Nanomechanical properties were measured using nanoindentation. Cell culture experiments were also conducted in order to ascertain the biocompatibility of the composite. The XRD results indicate that an intercalated structure was formed with an increase in d-spacing of montmorillonite. FTIR studies provide the evidence of molecular interaction among the three different constituents of the composite. AFM images show well-distributed nanoparticles in the chitosan matrix. The composites also exhibit a significant enhancement in nanomechanical property as compared to pure chitosan as well as the chi/HAP and chi/MMT composites. The TGA results indicate that an intercalated nanocomposite was formed with improved thermal properties even compared to chi/MMT composites. The results of cell culture experiments show that the composite is biocompatible and has a better cell proliferation rate compared to chi/HAP composites. This work represents the design of a novel clay-chitosan-hydroxyapatite composite with improved mechanical properties that has potential applications in bone tissue engineering.

  17. Characterization of thermoplastic polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by microcellular injection molding

    Energy Technology Data Exchange (ETDEWEB)

    Mi, Hao-Yang [National Engineering Research Center of Novel Equipment for Polymer Processing, South China University of Technology, Guangzhou (China); Department of Mechanical Engineering, University of Wisconsin–Madison, WI (United States); Salick, Max R. [Department of Engineering Physics, University of Wisconsin–Madison, WI (United States); Jing, Xin [National Engineering Research Center of Novel Equipment for Polymer Processing, South China University of Technology, Guangzhou (China); Department of Mechanical Engineering, University of Wisconsin–Madison, WI (United States); Jacques, Brianna R. [Department of Biology, University of Wisconsin–River Falls, WI (United States); Crone, Wendy C. [Department of Engineering Physics, University of Wisconsin–Madison, WI (United States); Peng, Xiang-Fang, E-mail: [National Engineering Research Center of Novel Equipment for Polymer Processing, South China University of Technology, Guangzhou (China); Turng, Lih-Sheng, E-mail: [Department of Mechanical Engineering, University of Wisconsin–Madison, WI (United States)


    Polylactic acid (PLA) and thermoplastic polyurethane (TPU) are two kinds of biocompatible and biodegradable polymers that can be used in biomedical applications. PLA has rigid mechanical properties while TPU possesses flexible mechanical properties. Blended TPU/PLA tissue engineering scaffolds at different ratios for tunable properties were fabricated via twin screw extrusion and microcellular injection molding techniques for the first time. Multiple test methods were used to characterize these materials. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of the two components in the blends; differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) confirmed the immiscibility between the TPU and PLA. Scanning electron microscopy (SEM) images verified that, at the composition ratios studied, PLA was dispersed as spheres or islands inside the TPU matrix and that this phase morphology further influenced the scaffold's microstructure and surface roughness. The blends exhibited a large range of mechanical properties that covered several human tissue requirements. 3T3 fibroblast cell culture showed that the scaffolds supported cell proliferation and migration properly. Most importantly, this study demonstrated the feasibility of mass producing biocompatible PLA/TPU scaffolds with tunable microstructures, surface roughnesses, and mechanical properties that have the potential to be used as artificial scaffolds in multiple tissue engineering applications. - Highlights: • Microcellular injection molding was used to fabricate tissue engineering scaffolds. • TPU/PLA tissue engineering scaffolds with tunable properties were fabricated. • Multiple test methods were used to characterize the scaffolds. • The biocompatibility of the scaffolds was confirmed by fibroblast cell culture. • Scaffolds produced have the potential to be used in multiple tissue applications.

  18. Characterization of a rediscovered haplosporidian parasite from cultured Penaeus vannamei. (United States)

    Nunan, Linda M; Lightner, Donald V; Pantoja, Carlos R; Stokes, Nancy A; Reece, Kimberly S


    Mortalities of Penaeus vannamei, cultured in ponds in Belize, Central America, began during the last part of the grow-out cycle during the cold weather months from September 2004 through February 2005. Tissue squashes of infected hepatopancreata and histological examination of infected shrimp revealed that the mortalities might have been caused by an endoparasite. To confirm the diagnosis, DNA was extracted from ethanol preserved hepatopancreata and the small-subunit rRNA gene was sequenced. The 1838 bp sequence was novel and phylogenetic analysis placed the P. vannamei parasite within the phylum Haplosporidia as a sister taxon to a clade that includes Bonamia and Minchinia species. In situ hybridization was performed using anti-sense DNA probes that were designed to hybridize specifically with the parasite's nucleic acid. This organism presents similar characteristics to those of a haplosporidian that infected cultured P. vannamei imported from Nicaragua into Cuba, as described by Dyková et al. (1988; Fish Dis 11:15-22).

  19. Turmeric powder (Curcuma longa Linn. as an antifungal agent in plant tissue culture studies

    Directory of Open Access Journals (Sweden)

    R.S. Upendra


    Full Text Available Culturing the individual plant cells, tissues (explants and organs in laboratory or in vitro on synthetic media (MS media under aseptic conditions is a usual process in plant tissue culture studies. The medium is rich in nutrients, also supports the growth of variety of microorganisms especially bacteria and fungi, which causecontamination of the medium, though the media is sterilized by autoclaving. During the process of cooling and transferring the media, the chances of fungal contamination remain high. This is avoided to the maximum extent following the good laboratory practices. A novel means could be incorporating turmeric, a well -known antifungal agent, into the media. In the present study, attempts were made to avoid fungal contamination using the media with various concentration of turmeric powder. Results of the investigation revealed that turmeric powder used at the concentrations of 0.8 g/L and 1.0 g/L in the media resulted in appreciable control of fungal contamination.

  20. Efficacy of resistance selection to Verticillium wilt in strawberry (Fragaria x ananassa Duch. tissue culture

    Directory of Open Access Journals (Sweden)

    Jadwiga I. Żebrowska


    Full Text Available The soil-borne pathogenic fungus Verticillium dahliae Kleb. causes economic losses in crops in temperate regions of the world and hence is the most studied species. Strawberry (Fragaria x ananassa Duch. belongs to plant species susceptible to Verticillium dahliae, although the response to infection caused by this pathogen is varied and depends on the cultivar. Due to a lack of efficient methods in Verticillium wilt elimination, the selection of genetically resistant plant material is a priority direction in breeding programs. Efficacy of resistance selection to Verticillium dahliae Kleb. in strawberry tissue culture was examined on the basis of response to in vitro infection by this pathogenic fungus in two tissue cultured strawberry cultivars, i.e. 'Filon' and 'Teresa'. Culture was conducted for 16 months in an environmentally controlled growth room at 18-20°C, 60-70% relative humidity and light intensity of 100 µm E × m-2 × s-1 on a 16h light / 8h dark cycle. Subcultures were proliferated every 6 weeks on modified Murashige and Skoog medium. Four hundred microplants from each tissue cultured cultivar were inoculated under in vitro conditions at the 4-leaf stage with a homogenate of liquid mycelium of Verticillium dahliae serving as the selecting agent. Disease symptoms were observed at 15, 30, 45, 60, and 75th days post inoculation. The extent of leaf chlorosis was rated on a scale of 0-4. At day 75th post inoculation, the percentage of totally chlorotic plants in micropropagated cv. Teresa reached the value of 76.27%, whereas the proportion of such plants in inoculated tissue cultured cv. Filon reached the value of 89.40%. Also, the index of infection calculated for very severe disease symptoms in the subclone 'Teresa' reached the mean value lower when compared with that calculated for subclone 'Filon' (0.0962 and 0.1150, respectively. These results suggested that the micropropagated cv. Teresa exhibited higher genetic resistance to the

  1. Multidimensional Monitoring of Keratin Intermediate Filaments in Cultured Cells and Tissues. (United States)

    Schwarz, Nicole; Moch, Marcin; Windoffer, Reinhard; Leube, Rudolf E


    Keratin filaments are a hallmark of epithelial differentiation. Their cell type-specific spatial organization and dynamic properties reflect and support epithelial function. To study this interdependency, imaging of fluorescently tagged keratins is a widely used method by which the temporospatial organization and behavior of the keratin intermediate filament network can be analyzed in living cells. Here, we describe methods that have been adapted and optimized to dissect and quantify keratin intermediate filament network dynamics in vital cultured cells and functional tissues.

  2. Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures (United States)

    Muñoz-Muñoz, Ana Celia; Gutiérrez-Pulido, Humberto; Rodríguez-Domínguez, José Manuel; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín; Cervantes-Martínez, Jesús


    We demonstrate the effectiveness of laser-induced fluorescence (LIF) for monitoring the development and stress detection of in vitro tissue cultures in a nondestructive and noninvasive way. The changes in LIF spectra caused by the induction of organogenesis, the increase of the F690/F740 ratio as a result of the stress originated in the organogenic explants due to shoot emergence, and the relationship between fluorescence spectra and shoot development were detected by LIF through closed containers of Saintpaulia ionantha.

  3. Characterization of culturable bacteria isolated from the cold-water coral Lophelia pertusa (United States)

    Galkiewicz, Julia P.; Pratte, Zoe A.; Gray, Michael A.; Kellogg, Christina A.


    Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial-coral associations. The Kirby-Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.

  4. Culture and characterization of mammary cancer stem cells in mammospheres. (United States)

    Piscitelli, Eleonora; Cocola, Cinzia; Thaden, Frank Rüdiger; Pelucchi, Paride; Gray, Brian; Bertalot, Giovanni; Albertini, Alberto; Reinbold, Rolland; Zucchi, Ileana


    Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation.

  5. Polarization-sensitive multispectral tissue characterization for optimizing intestinal anastomosis (United States)

    Cha, Jaepyeong; Triana, Brian; Shademan, Azad; Krieger, Axel; Kim, Peter C. W.; Kang, Jin U.


    A novel imaging system that recommends potential suture placement for anastomosis to surgeons is developed. This is achieved by a multispectral imaging system coupled with polarizers and image analysis software. We performed preliminary imaging of ex vivo porcine intestine to evaluate the system. Vulnerable tissue regions including blood vessels were successfully identified and segmented. Thickness of different tissue areas is visualized. Strategies towards optimal points for suture placements have been discussed. Preliminary data suggest our imaging platform and analysis algorithm may be useful in avoiding blood vessels, identifying optimal regions for suture placements to perform safer operations in possibly reduced time.

  6. Mechanical Stimulus Inhibits the Growth of a Bone Tissue Model Cultured In Vitro

    Institute of Scientific and Technical Information of China (English)

    Zong-ming Wan; Lu Liu; Jian-yu Li; Rui-xin Li; Yong Guo; Hao Li; Jian-ming Zhang; Xi-zheng Zhang


    Objectives To construct the cancellous bone explant model and a method of culturing these bone tissues in vitro, and to investigate the effect of mechanical load on growth of cancellous bone tissue in vitro. Methods Cancellous bone were extracted from rabbit femoral head and cut into 1-mm-thick and 8-mm-diameter slices under sterile conditions. HE staining and scanning electron microscopy were employed to identify the histomorphology of the model after being cultured with a new dynamic load and circulating perfusion bioreactor system for 0, 3, 5, and 7 days, respectively. We built a three-dimensional model using microCT and analyzed the loading effects using finite element analysis. The model was subjected to mechanical load of 1000, 2000, 3000, and 4000μεrespectively for 30 minutes per day. After 5 days of continuous stimuli, the activities of alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (TRAP) were detected. Apoptosis was analyzed by DNA ladder detection and caspase-3/8/9 activity detection. Results After being cultured for 3, 5, and 7 days, the bone explant model grew well. HE staining showed the apparent nucleus in cells at the each indicated time, and electron microscope revealed the living cells in the bone tissue. The activities of AKP and TRAP in the bone explant model under mechanical load of 3000 and 4000μεwere significantly lower than those in the unstressed bone tissues (all P Conclusions The cancellous bone explant model extracted from the rabbit femoral head could be alive at least for 7 days in the dynamic load and circulating perfusion bioreactor system, however, pathological mechanical load could affect the bone tissue growth by apoptosis in vitro. The differentiation of osteoblasts and osteoclasts might be inhibited after the model is stimulated by mechanical load of 3000 and 4000με.

  7. Toxicity and oxidative stress of canine mesenchymal stromal cells from adipose tissue in different culture passages

    Directory of Open Access Journals (Sweden)

    Arícia Gomes Sprada


    Full Text Available Abstract: Stem cells in regenerative therapy have received attention from researchers in recent decades. The culture of these cells allows studies about their behavior and metabolism. Thus, cell culture is the basis for cell therapy and tissue engineering researches. A major concern regarding the use of cultivated stem cell in human or veterinary clinical routine is the risk of carcinogenesis. Cellular activities require a balanced redox state. However, when there is an imbalance in this state, oxidative stress occurs. Oxidative stress contributes to cytotoxicity, which may result in cell death or genomic alterations, favoring the development of cancer cells. The aim of this study was to determine whether there are differences in the behavior of cultured mesenchymal stem cells from canine adipose tissue according to its site of collection (omentum and subcutaneous evaluating the rate of proliferation, viability, level of oxidative stress and cytotoxicity over six passages. For this experiment, two samples of adipose tissue from subcutaneous and omentum where taken from a female dog corpse, 13 years old, Pitbull. The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death.

  8. Identification of neurotoxic cytokines by profiling Alzheimer's disease tissues and neuron culture viability screening. (United States)

    Wood, Levi B; Winslow, Ashley R; Proctor, Elizabeth A; McGuone, Declan; Mordes, Daniel A; Frosch, Matthew P; Hyman, Bradley T; Lauffenburger, Douglas A; Haigis, Kevin M


    Alzheimer's disease (AD) therapeutics based on the amyloid hypothesis have shown minimal efficacy in patients, suggesting that the activity of amyloid beta (Aβ) represents only one aspect of AD pathogenesis. Since neuroinflammation is thought to play an important role in AD, we hypothesized that cytokines may play a direct role in promoting neuronal death. Here, we profiled cytokine expression in a small cohort of human AD and control brain tissues. We identified AD-associated cytokines using partial least squares regression to correlate cytokine expression with quantified pathologic disease state and then used neuron cultures to test whether cytokines up-regulated in AD tissues could affect neuronal viability. This analysis identified cytokines that were associated with the pathological severity. Of the top correlates, only TNF-α reduced viability in neuron culture when applied alone. VEGF also reduced viability when applied together with Aβ, which was surprising because VEGF has been viewed as a neuro-protective protein. We found that this synthetic pro-death effect of VEGF in the context of Aβ was commensurate with VEGFR-dependent changes in multiple signaling pathways that govern cell fate. Our findings suggest that profiling of tissues combined with a culture-based screening approach can successfully identify new mechanisms driving neuronal death.

  9. Biodynamic Doppler imaging of subcellular motion inside 3D living tissue culture and biopsies (Conference Presentation) (United States)

    Nolte, David D.


    Biodynamic imaging is an emerging 3D optical imaging technology that probes up to 1 mm deep inside three-dimensional living tissue using short-coherence dynamic light scattering to measure the intracellular motions of cells inside their natural microenvironments. Biodynamic imaging is label-free and non-invasive. The information content of biodynamic imaging is captured through tissue dynamics spectroscopy that displays the changes in the Doppler signatures from intracellular constituents in response to applied compounds. The affected dynamic intracellular mechanisms include organelle transport, membrane undulations, cytoskeletal restructuring, strain at cellular adhesions, cytokinesis, mitosis, exo- and endo-cytosis among others. The development of 3D high-content assays such as biodynamic profiling can become a critical new tool for assessing efficacy of drugs and the suitability of specific types of tissue growth for drug discovery and development. The use of biodynamic profiling to predict clinical outcome of living biopsies to cancer therapeutics can be developed into a phenotypic companion diagnostic, as well as a new tool for therapy selection in personalized medicine. This invited talk will present an overview of the optical, physical and physiological processes involved in biodynamic imaging. Several different biodynamic imaging modalities include motility contrast imaging (MCI), tissue-dynamics spectroscopy (TDS) and tissue-dynamics imaging (TDI). A wide range of potential applications will be described that include process monitoring for 3D tissue culture, drug discovery and development, cancer therapy selection, embryo assessment for in-vitro fertilization and artificial reproductive technologies, among others.

  10. Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer myostatin gene

    Directory of Open Access Journals (Sweden)

    Smith-Keune Carolyn


    Full Text Available Abstract Background Myostatin (MSTN is a member of the transforming growth factor-β superfamily that negatively regulates growth of skeletal muscle tissue. The gene encoding for the MSTN peptide is a consolidate candidate for the enhancement of productivity in terrestrial livestock. This gene potentially represents an important target for growth improvement of cultured finfish. Results Here we report molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer MSTN-1 gene. The barramundi MSTN-1 was encoded by three exons 379, 371 and 381 bp in length and translated into a 376-amino acid peptide. Intron 1 and 2 were 412 and 819 bp in length and presented typical GT...AG splicing sites. The upstream region contained cis-regulatory elements such as TATA-box and E-boxes. A first assessment of sequence variability suggested that higher mutation rates are found in the 5' flanking region with several SNP's present in this species. A putative micro RNA target site has also been observed in the 3'UTR (untranslated region and is highly conserved across teleost fish. The deduced amino acid sequence was conserved across vertebrates and exhibited characteristic conserved putative functional residues including a cleavage motif of proteolysis (RXXR, nine cysteines and two glycosilation sites. A qualitative analysis of the barramundi MSTN-1 expression pattern revealed that, in adult fish, transcripts are differentially expressed in various tissues other than skeletal muscles including gill, heart, kidney, intestine, liver, spleen, eye, gonad and brain. Conclusion Our findings provide valuable insights such as sequence variation and genomic information which will aid the further investigation of the barramundi MSTN-1 gene in association with growth. The finding for the first time in finfish MSTN of a miRNA target site in the 3'UTR provides an opportunity for the identification of regulatory mutations on the

  11. An evolutionary view of plant tissue culture: somaclonal variation and selection. (United States)

    Wang, Qin-Mei; Wang, Li


    Plants regenerated from in vitro cultures possess an array of genetic and epigenetic changes. This phenomenon is known as 'somaclonal variation' and the frequency of somaclonal variation (SV) is usually elevated far beyond that expected in nature. Initially, the relationship between time in culture and detected SV was found to support the widespread belief that SV accumulates with culture age. However, a few studies indicated that older cultures yielded regenerants with less SV. What leads to this seemed contradiction? In this article, we have proposed a novel in vitro callus selection hypothesis, differentiation bottleneck (D-bottleneck) and dedifferentiation bottleneck (Dd-bottleneck), which consider natural selection theory to be fit for cell population in vitro. The results of multiplication races between the cells with the true-to-type phenotype and the deleterious cells determine the increase/decrease of SV frequencies in calli or regenerants as in vitro culture time goes on. The possibility of interpreting the complex situation of time-related SV by the evolutionary theory is discussed in this paper. In addition, the SV threshold, space-determined hypothesis and D-bottleneck are proposed to interpret the loss of the regenerability through a long period of plant tissue culture (PTC).

  12. Multispectral characterization of tissues encountered during laparoscopic colorectal surgery

    NARCIS (Netherlands)

    Schols, R.M.; Dunias, P.; Wieringa, F.P.; Stassen, L.P.


    Aims: This study investigated the feasibility of automated differentiation between essential tissue types encountered during laparoscopic colorectal surgery using spectral analysis. Methods: Wide band (440-1830 nm) spectra were collected using an optical fiber probe and spectrometer from freshly exp

  13. The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content. (United States)

    Delain, E; Aouani, A; Vignal, A; Couture-Tosi, E; Hovsépian, S; Fayet, G


    The major protein synthesized in vitro by the ovine thyroid cell line OVNIS 6H is the prothyroid hormone thyroglobulin. Purified from serum-free cell culture media using sucrose gradient centrifugation, the thyroglobulin dimer was analysed for iodine content and observed by electron microscopy. In their usual medium, the OVNIS 6H cells produce a very poorly iodinated thyroglobulin containing 0.05 I atom per molecule. When cultured with methimazole or propylthiouracil, two inhibitors of iodide organification, less than 0.007 I atom/molecules was found. These molecules purified from cell cultures were compared to those purified from ovine thyroid tissue containing 26 I atoms/mol. Despite large differences in iodine content, the three preparations all consist of 19 S thyroglobulin dimers with the classical ovoidal shape. The variability in size measurements remains in a 2% range for all thyroglobulin types. Consequently, no real significant variation can be found between the highly iodinated thyroglobulin isolated from tissue, and the poorly or non-iodinated thyroglobulins isolated from cells cultured with or without methimazole or propylthiouracil.

  14. 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.

  15. Characterization of primary and secondary cultures of astrocytes prepared from mouse cerebral cortex

    DEFF Research Database (Denmark)

    Skytt, Dorte Marie; Madsen, Karsten Kirkegaard; Pajecka, Kamilla;


    Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary ac...

  16. Proteomic characterization of adipose tissue constituents, a necessary step for understanding adipose tissue complexity. (United States)

    Peinado, Juan R; Pardo, María; de la Rosa, Olga; Malagón, Maria M


    The original concept of adipose tissue as an inert storage depot for the excess of energy has evolved over the last years and it is now considered as one of the most important organs regulating body homeostasis. This conceptual change has been supported by the demonstration that adipose tissue serves as a major endocrine organ, producing a wide variety of bioactive molecules, collectively termed adipokines, with endocrine, paracrine and autocrine activities. Adipose tissue is indeed a complex organ wherein mature adipocytes coexist with the various cell types comprising the stromal-vascular fraction (SVF), including preadipocytes, adipose-derived stem cells, perivascular cells, and blood cells. It is known that not only mature adipocytes but also the components of SVF produce adipokines. Furthermore, adipokine production, proliferative and metabolic activities and response to regulatory signals (i.e. insulin, catecholamines) differ between the different fat depots, which have been proposed to underlie their distinct association to specific diseases. Herein, we discuss the recent proteomic studies on adipose tissue focused on the analysis of the separate cellular components and their secretory products, with the aim of identifying the basic features and the contribution of each component to different adipose tissue-associated pathologies.

  17. Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

    Directory of Open Access Journals (Sweden)

    Lin Wang

    Full Text Available BACKGROUND: The basic strategy to construct tissue engineered bone graft (TEBG is to combine osteoblastic cells with three dimensional (3D scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. METHODS: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP scaffold fabricated by Rapid Prototyping (RP technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC method, static seeding and perfusion culture (SSPC method, and static seeding and static culture (SSSC method for their in vitro performance and bone defect healing efficacy with a rabbit model. RESULTS: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. CONCLUSION: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and

  18. Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea. (United States)

    Kenta, Tanaka; Edwards, Jessica E M; Butlin, Roger K; Burke, Terry; Quick, W Paul; Urwin, Peter; Davey, Matthew P


    While genotype-environment interaction is increasingly receiving attention by ecologists and evolutionary biologists, such studies need genetically homogeneous replicates-a challenging hurdle in outcrossing plants. This could be potentially overcome by using tissue culture techniques. However, plants regenerated from tissue culture may show aberrant phenotypes and "somaclonal" variation. Here, we examined somaclonal variation due to tissue culturing using the response to cold treatment of photosynthetic efficiency (chlorophyll fluorescence measurements for Fv/Fm, Fv'/Fm', and ΦPSII, representing maximum efficiency of photosynthesis for dark- and light-adapted leaves, and the actual electron transport operating efficiency, respectively, which are reliable indicators of photoinhibition and damage to the photosynthetic electron transport system). We compared this to variation among half-sibling seedlings from three different families of Arabidopsis lyrata ssp. petraea Somaclonal variation was limited, and we could detect within-family variation in change in chlorophyll fluorescence due to cold shock successfully with the help of tissue-culture derived replicates. Icelandic and Norwegian families exhibited higher chlorophyll fluorescence, suggesting higher performance after cold shock, than a Swedish family. Although the main effect of tissue culture on Fv/Fm, Fv'/Fm', and ΦPSII was small, there were significant interactions between tissue culture and family, suggesting that the effect of tissue culture is genotype-specific. Tissue-cultured plantlets were less affected by cold treatment than seedlings, but to a different extent in each family. These interactive effects, however, were comparable to, or much smaller than the single effect of family. These results suggest that tissue culture is a useful method for obtaining genetically homogenous replicates for studying genotype-environment interaction related to adaptively-relevant phenotypes, such as cold response, in

  19. The use of the compound probability density function in ultrasonic tissue characterization

    Energy Technology Data Exchange (ETDEWEB)

    Shankar, P M [Department of Electrical and Computer Engineering, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States)


    Recently, a compound probability density function (pdf) was proposed to model the envelope of the ultrasonic backscattered echo from tissues. This pdf will allow local and global variations in scattering cross sections and even multiple scattering in tissue. It approximates to the Nakagami, K or Rayleigh distributions under different limiting conditions, thus making it very versatile. In this work, a new parameter associated with compound pdf, speckle factor, has been introduced to characterize the scattering conditions. The usefulness of this parameter for tissue characterization has been explored through computer simulation of ultrasonic A scans and analyses of the data collected from tissue-mimicking phantoms. Results suggest the potential applications of the compound pdf and its parameters in ultrasonic tissue characterization.

  20. Leaf and root volatiles produced by tissue cultures of Alpinia zerumbet (pers. Burtt & Smith under the influence of different plant growth regulators

    Directory of Open Access Journals (Sweden)

    Cristiane Pimentel Victório


    Full Text Available Volatiles produced by plantlets of Alpinia zerumbet were obtained by means of simultaneous distillation-extraction (SDE. The effects of indole-3-acetic acid, kinetin, thidiazuron and 6-benzylaminopurine on leaf and root volatile composition obtained by tissue cultures were investigated. A higher content of b-pinene and a lower content of sabinene were observed in leaf volatile of plantlets cultured in control, IAA and IAA+ TDZ media, as compared with those of donor plants. In vitro conditions were favorable to increase caryophyllene content. Volatile compounds from the root were characterized mainly by camphene, fenchyl-acetate and bornyl acetate; which constitute about 60% of total volatile.

  1. Characterizing microscopic domains of birefringence in thin tissue sections (United States)

    Jacques, Steven L.; Moody, Alex; Ramella-Roman, Jessica C.


    A tissue engineering problem that we anticipate will become increasingly of interest is how to grow protein layers and filaments in preferred orientations. For example, the polymerization of monomers into an oriented structure which may exert influence on adherent cells. In this paper, we report on an optical solution using polarized light measurements to probe the structure and orientation of fibers. In particular in this initial study, we measure the fast-axis orientation and retardance of micro-domains in thin sections of liver, muscle, and skin tissues using a polarizing microscope. The size of microdomains of iso- retardance is in the range 10-100 μm, which suggests that optical measurements with laser beams that are on the order of 1-mm in diameter or with imaging cameras with pixels sizes on the order of 100 s of μm will average over several microdomains and consequently complicate interpretation of measurements.

  2. Tissue specific metal characterization of selected fish species in Pakistan. (United States)

    Ahmed, Mukhtiar; Ahmad, Taufiq; Liaquat, Muhammad; Abbasi, Kashif Sarfraz; Farid, Ibrahim Bayoumi Abdel; Jahangir, Muhammad


    Concentration of various metals, i.e., zinc (Zn), copper (Cu), lead (Pb), nickel (Ni), iron (Fe), manganese (Mn), chromium (Cr), and silver (Ag), was evaluated in five indigenous fish species (namely, silver carp, common carp, mahseer, thela fish, and rainbow trout), by using atomic absorption spectrophotometer. It is proved from this study that, overall, mahseer and rainbow trout had high amount of zinc, whereas thela fish and silver carp had high concentration of copper, chromium, silver, nickel, and lead, while common carp had highest amount of iron contents. Furthermore, a tissue-specific discrimination among various fish species was observed, where higher metal concentrations were noticed in fish liver, with decreasing concentration in other organs like skin, gills, and finally the least contents in fish muscle. Multivariate data analysis showed not only a variation in heavy metals among the tissues but also discrimination among the selected fish species.

  3. Suction based mechanical characterization of superficial facial soft tissues. (United States)

    Weickenmeier, J; Jabareen, M; Mazza, E


    The present study is aimed at a combined experimental and numerical investigation of the mechanical response of superficial facial tissues. Suction based experiments provide the location, time, and history dependent behavior of skin and SMAS (superficial musculoaponeurotic system) by means of Cutometer and Aspiration measurements. The suction method is particularly suitable for in vivo, multi-axial testing of soft biological tissue including a high repeatability in subsequent tests. The campaign comprises three measurement sites in the face, i.e. jaw, parotid, and forehead, using two different loading profiles (instantaneous loading and a linearly increasing and decreasing loading curve), multiple loading magnitudes, and cyclic loading cases to quantify history dependent behavior. In an inverse finite element analysis based on anatomically detailed models an optimized set of material parameters for the implementation of an elastic-viscoplastic material model was determined, yielding an initial shear modulus of 2.32kPa for skin and 0.05kPa for SMAS, respectively. Apex displacements at maximum instantaneous and linear loading showed significant location specificity with variations of up to 18% with respect to the facial average response while observing variations in repeated measurements in the same location of less than 12%. In summary, the proposed parameter sets for skin and SMAS are shown to provide remarkable agreement between the experimentally observed and numerically predicted tissue response under all loading conditions considered in the present study, including cyclic tests.

  4. Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine. (United States)

    Rana, Deepti; Ramasamy, Keerthana; Leena, Maria; Jiménez, Constanza; Campos, Javier; Ibarra, Paula; Haidar, Ziyad S; Ramalingam, Murugan


    Stem cell-based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial-based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell-based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554-567, 2016.

  5. Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH). (United States)

    Stępińska, M; Olszewska-Sosińska, O; Lau-Dworak, M; Zielnik-Jurkiewicz, B; Trafny, E A


    Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

  6. Use of stationary focused ultrasound fields for characterization of tissue and localized tissue ablation (United States)

    Winey, Brian Andrew

    Ultrasound-induced blood stasis has been observed for more than 30 years. The physical understanding of the phenomenon has not been fully explored. Analytical descriptions of the acoustic interaction with spheres in suspension have been derived but the physical implications and limitations have not been demonstrated. The analytical expressions will be tested against physical observations using numerical simulations. The simulations will begin with stationary spheres and continue with the inclusion of moving spheres and a moving suspending fluid. To date, experimental observations of acoustically induced blood stasis have been either in vitro or invasive. We demonstrate ultrasound-induced blood stasis in murine normal leg muscle versus tumor-bearing legs, observed through noninvasive measurements of optical spectroscopy, and discuss possible diagnostic uses for this effect of ultrasound. We derive the optimal optical wavelengths for measuring the effects of the ultrasound at small source detector separations. Using optical oximetry performed at the optimal wavelengths, we demonstrate that effects of ultrasound can be used to differentiate tumor from normal leg muscle tissue in mice. To provide a statistical analysis of the experiments, we propose a novel diagnostic algorithm that quantitatively differentiates tumor from nontumor with maximum specificity 0.83, maximum sensitivity 0.79, and area under receiver-operating-characteristics curve 0.90. Ultrasound has long been known to cause tissue heating when applied in high intensities. More recently, interest has arisen in the area of High Intensity Focused Ultrasound (HIFU) for localized tissue heating effects, specifically thermal ablation. All present techniques employ focused traveling high intensity acoustic waves to create a region of elevated temperature. Such high intensity traveling waves can be damaging to normal tissue in the vicinity of the focal region, and have demonstrated surface burns and caused

  7. Breast phantom for mammary tissue characterization by near infrared spectroscopy (United States)

    Miranda, D. A.; Cristiano, K. L.; Gutiérrez, J. C.


    Breast cancer is a disease associated to a high morbidity and mortality in the entire world. In the study of early detection of breast cancer the development of phantom is so important. In this research we fabricate a breast phantom using a ballistic gel with special modifications to simulate a normal and abnormal human breast. Optical properties of woman breast in the near infrared region were modelled with the phantom we developed. The developed phantom was evaluated with near infrared spectroscopy in order to study its relation with breast tissue. A good optical behaviour was achieved with the model fabricated.

  8. Enhancing plant regeneration in tissue culture: a molecular approach through manipulation of cytokinin sensitivity. (United States)

    Hill, Kristine; Schaller, G Eric


    Micropropagation is used for commercial purposes worldwide, but the capacity to undergo somatic organogenesis and plant regeneration varies greatly among species. The plant hormones auxin and cytokinin are critical for plant regeneration in tissue culture, with cytokinin playing an instrumental role in shoot organogenesis. Type-B response regulators govern the transcriptional output in response to cytokinin and are required for plant regeneration. In our paper published in Plant Physiology, we explored the functional redundancy among the 11 type-B Arabidopsis response regulators (ARRs). Interestingly, we discovered that the enhanced expression of one family member, ARR10, induced hypersensitivity to cytokinin in multiple assays, including callus greening and shoot induction of explants. Here we 1) discuss the hormone dependence for in vitro plant regeneration, 2) how manipulation of the cytokinin response has been used to enhance plant regeneration, and 3) the potential of the ARR10 transgene as a tool to increase the regeneration capacity of agriculturally important crop plants. The efficacy of ARR10 for enhancing plant regeneration likely arises from its ability to transcriptionally regulate key cytokinin responsive genes combined with an enhanced protein stability of ARR10 compared with other type-B ARRs. By increasing the capacity of key tissues and cell types to respond to cytokinin, ARR10, or other type-B response regulators with similar properties, could be used as a tool to combat the recalcitrance of some crop species to tissue culture techniques.

  9. Targeted metabolomics in cultured cells and tissues by mass spectrometry: method development and validation. (United States)

    Abdel Rahman, Anas M; Pawling, Judy; Ryczko, Michael; Caudy, Amy A; Dennis, James W


    Metabolomics is the identification and quantitation of small bio-molecules (metabolites) in biological samples under various environmental and genetic conditions. Mass spectrometry provides the unique opportunity for targeted identification and quantification of known metabolites by selective reaction monitoring (SRM). However, reproducibility of this approach depends on careful consideration of sample preparation, chemical classes, and stability of metabolites to be evaluated. Herein, we introduce and validate a targeted metabolite profiling workflow for cultured cells and tissues by liquid chromatography-triple quadrupole tandem mass spectrometry. The method requires a one-step extraction of water-soluble metabolites and targeted analysis of central metabolites that include glycolysis, amino acids, nucleotides, citric acid cycle, and the hexosamine biosynthetic pathway. The sensitivity, reproducibility and molecular stability of each targeted metabolite were assessed under experimental conditions. Quantitation of metabolites by peak area ratio was linear with a dilution over a 4 fold dynamic range with minimal deviation R(2)=0.98. Inter- and intra-day precision with cells and tissues had an average coefficient of variation <15% for cultured cell lines, and somewhat higher for mouse liver tissues. The method applied in triplicate measurements readily distinguished immortalized cells from malignant cells, as well as mouse littermates based on their hepatic metabolic profiles.

  10. Implementing oxygen control in chip-based cell and tissue culture systems. (United States)

    Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth


    Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

  11. Characterization of various tissue mimicking materials for medical ultrasound imaging (United States)

    Thouvenot, Audrey; Poepping, Tamie; Peters, Terry M.; Chen, Elvis C. S.


    Tissue mimicking materials are physical constructs exhibiting certain desired properties, which are used in machine calibration, medical imaging research, surgical planning, training, and simulation. For medical ultrasound, those specific properties include acoustic propagation speed and attenuation coefficient over the diagnostic frequency range. We investigated the acoustic characteristics of polyvinyl chloride (PVC) plastisol, polydimethylsiloxane (PDMS), and isopropanol using a time-of-light technique, where a pulse was passed through a sample of known thickness contained in a water bath. The propagation speed in PVC is approximately 1400ms-1 depending on the exact chemical composition, with the attenuation coefficient ranging from 0:35 dB cm-1 at 1MHz to 10:57 dB cm-1 at 9 MHz. The propagation speed in PDMS is in the range of 1100ms-1, with an attenuation coefficient of 1:28 dB cm-1 at 1MHz to 21:22 dB cm-1 at 9 MHz. At room temperature (22 °C), a mixture of water-isopropanol (7:25% isopropanol by volume) exhibits a propagation speed of 1540ms-1, making it an excellent and inexpensive tissue-mimicking liquid for medical ultrasound imaging.

  12. Characterization of Heparin-binding Site of Tissue Transglutaminase (United States)

    Wang, Zhuo; Collighan, Russell J.; Pytel, Kamila; Rathbone, Daniel L.; Li, Xiaoling; Griffin, Martin


    Tissue transglutaminase (TG2) is a multifunctional Ca2+-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed. PMID:22298777

  13. Fabrication and characterization of bio-engineered cardiac pseudo tissues

    Energy Technology Data Exchange (ETDEWEB)

    Xu Tao; Boland, Thomas [Department of Bioengineering, 420 Rhodes Hall, Clemson University, Clemson, SC 29634 (United States); Baicu, Catalin; Aho, Michael; Zile, Michael, E-mail: tboland@clemson.ed [Department of Medicine, Medical University of South Carolina, Charleston, SC 29425 (United States)


    We report on fabricating functional three-dimensional (3D) tissue constructs using an inkjet based bio-prototyping method. With the use of modified inkjet printers, contractile cardiac hybrids that exhibit the forms of the 3D rectangular sheet and even the 'half heart' (with two connected ventricles) have been fabricated by arranging alternate layers of biocompatible alginate hydrogels and mammalian cardiac cells according to pre-designed 3D patterns. In this study, primary feline adult and H1 cardiomyocytes were used as model cardiac cells. Alginate hydrogels with controlled micro-shell structures were built by spraying cross-linkers in micro-drops onto un-gelled alginic acid. The cells remained viable in constructs as thick as 1 cm due to the programmed porosity. Microscopic and macroscopic contractile functions of these cardiomyocyte constructs were observed in vitro. These results suggest that the inkjet bio-prototyping method could be used for hierarchical design of functional cardiac pseudo tissues, balanced with porosity for mass transport and structural support.

  14. In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Dao-Cai [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Department of Stomatology, The 291st Hospital of P.L.A, Baotou (China); Li, De-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ji, Hui-Cang [Military Sanatorium of Retired Cadres, Baotou (China); Rao, Guo-Zhou [Center of Laboratory, School of Stomatology, Xi' an Jiaotong University, Xi' an (China); Liang, Li-Hua [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China); Ma, Ai-Jie [Xi' an Technology University, Xi' an (China); Xie, Chao; Zou, Gui-Ke; Song, Ying-Liang [Department of Implant Dentistry, School of Stomatology, Fourth Military Medical University, Xi' an (China)


    In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

  15. Characterization of Absorbent Flow Rate in Towel and Tissue

    Directory of Open Access Journals (Sweden)

    Paul D. Beuther


    Full Text Available The quality of a paper towel is often judged based on how quickly it can wipe up a spill. However, the test methods currently available cannot repeatably measure significant differences in absorbent rate between samples. Recent round-robin testing evaluations by TAPPI and CEN organizations have shown that past methods, such as ASTM D5802-95 and TAPPI T561-pm [1], are unreliable due to high variability. The reasons for the lack of repeatability are unclear. The relation between the wicking mechanism and the fundamental absorbent properties needs to be better understood. This paper uses x-ray imaging to show the overall flow characteristics of fluid absorption within a towel, and compares the results to model predictions to show which parameters are important to the process. From this understanding, a revised test method is proposed that provides adequate statistical discernment of absorbent rate properties of tissue on a simple lab-bench scale device.

  16. Characterizing microstructure of living tissues with time-dependent diffusion

    CERN Document Server

    Novikov, Dmitry S; Jensen, Jens H; Helpern, Joseph A


    Molecular diffusion measurements are widely used to probe microstructure in materials and living organisms noninvasively. The precise relation of diffusion metrics to microstructure remains a major challenge: In complex samples, it is often unclear which structural features are most relevant and can be quantified. Here we classify the structural complexity in terms of the long time tail exponent in the molecular velocity autocorrelation function. The specific values of the dynamical exponent let us identify the relevant tissue microanatomy affecting water diffusion measured with MRI in muscles and in brain, and the microstructural changes in ischemic stroke. Our framework presents a systematic way to identify the most relevant part of structural complexity using transport measured with a variety of techniques.

  17. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses. (United States)

    Ozbun, Michelle A; Patterson, Nicole A


    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

  18. Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering. (United States)

    Deng, Cheri X; Hong, Xiaowei; Stegemann, Jan P


    Ultrasound techniques are increasingly being used to quantitatively characterize both native and engineered tissues. This review provides an overview and selected examples of the main techniques used in these applications. Grayscale imaging has been used to characterize extracellular matrix deposition, and quantitative ultrasound imaging based on the integrated backscatter coefficient has been applied to estimating cell concentrations and matrix morphology in tissue engineering. Spectral analysis has been employed to characterize the concentration and spatial distribution of mineral particles in a construct, as well as to monitor mineral deposition by cells over time. Ultrasound techniques have also been used to measure the mechanical properties of native and engineered tissues. Conventional ultrasound elasticity imaging and acoustic radiation force imaging have been applied to detect regions of altered stiffness within tissues. Sonorheometry and monitoring of steady-state excitation and recovery have been used to characterize viscoelastic properties of tissue using a single transducer to both deform and image the sample. Dual-mode ultrasound elastography uses separate ultrasound transducers to produce a more potent deformation force to microscale characterization of viscoelasticity of hydrogel constructs. These ultrasound-based techniques have high potential to impact the field of tissue engineering as they are further developed and their range of applications expands.

  19. Effect of interferon on the development of Trypanosoma cruzi in tissue culture "Vero" cells

    Directory of Open Access Journals (Sweden)

    R. R. Golgher


    Full Text Available Results are presented on the effects of interferon on the intracellular stages of T. cruzi in tissue culture "Vero" cells. Interferon was obtained by infecting monolayers of human amniotic cells with inactivated Newcastle disease virus. Interferon has not affected the cell infection by T. cruzi culture infective stages and neither has it prevented the transformation of amastigote into trypomastigote stages.Interferon obtido através da infecção de células amnióticas humanas por vírus inativado da doença de Newcastle foi incapaz de influir sobre a infectividade de formas de cultura do T. cruzi para células "Vero" de cultura de tecido. A transformação amastigota-tripomastigota também não foi afetada pelo interferon.

  20. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia. (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A


    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  1. The safety assessment of food ingredients derived from plant cell, tissue and organ cultures: a review. (United States)

    Murthy, Hosakatte Niranjana; Georgiev, Milen I; Park, So-Young; Dandin, Vijayalaxmi S; Paek, Kee-Yoeup


    Plant cell, tissue and organ cultures (PCTOC) have become an increasingly attractive alternative for the production of various high molecular weight molecules which are used as flavourings, fragrances, colouring agents and food additives. Although PCTOC products are cultivated in vitro in a contamination free environment, the raw material produced from PCTOC may contain many components apart from the target compound. In some cases, PCTOC raw materials may also carry toxins, which may be naturally occurring or accumulated during the culture process. Assessment of the safety of PCTOC products is, therefore, a priority of the biotech industries involved in their production. The safety assessment involves the evaluation of starting material, production process and the end product. Before commercialisation, PCTOC products should be evaluated for their chemical and biological properties, as well as for their toxicity. In this review, measures and general criteria for biosafety evaluation of PCTOC products are addressed and thoroughly discussed.


    Directory of Open Access Journals (Sweden)



    Full Text Available Artificially inoculated asparagus tissue culture plantlets with a virulent fungus, Fusarium proliferatum showed signs of infection as early as 4 days after inoculat ion. Macroscopic observations revealed presence of early symptoms such as necrotic lesions at the affected area and light microscopic examinations clearly revealed the post-penetration events that took place including the destruction of surrounding cells. However, little is known of the hyphal activity or advancement on the host's surface at the initial stage after inoculation. Scanning electron microscopic examination clearly revealed the hyphal advancement on the surface and the mode of entrance into the host tissues beneath. Four days after inoculation, the fungi proceeded to spread out from the inoculation point onto the host surface which eventually developed into a sparse network of both aerial and non-aerial hyphae. Non-aerial hyphae form a network of mycelium that adheres to the surface and it's movement appeared to be oriented towards the stomata. Hyphal penetration occurs more often through the stomata, natural openings or wounds. In some cases, the hyphae crossed over the stomatal opening w ithout entering the host tissues. At places where the cuticle layer is absent or not well developed the hyphae successfully grew in between the epidermal cells into the tissues beneath.

  3. Target detect system in 3D using vision apply on plant reproduction by tissue culture (United States)

    Vazquez Rueda, Martin G.; Hahn, Federico


    This paper presents the preliminary results for a system in tree dimension that use a system vision to manipulate plants in a tissue culture process. The system is able to estimate the position of the plant in the work area, first calculate the position and send information to the mechanical system, and recalculate the position again, and if it is necessary, repositioning the mechanical system, using an neural system to improve the location of the plant. The system use only the system vision to sense the position and control loop using a neural system to detect the target and positioning the mechanical system, the results are compared with an open loop system.


    Directory of Open Access Journals (Sweden)

    V. A. Sidorov


    Full Text Available Plant genetic transformation has become an important biotechnology tool for the improvement of many crops. A solid foundation for the fast development and implementation of biotechnology in agriculture was provided by achievements in plant tissue culture. On the 30th anniversary of plant transformation, I report the advancements, recent challenges and shifts in methodology of transformation. The main focus of this paper will be on conventional and novel approaches for genetic improvements of soybean, cotton and corn. I will also highlight results on the transformation of these crops that have considerably been improved by modern biotechnology.

  5. Tissue culture study of the medicinal plant leek (allium ampeloprasum L). (United States)

    Monemi, Mohammad Bagher; Kazemitabar, S Kamal; Bakhshee Khaniki, Gholamreza; Yasari, Esmaeil; Sohrevardi, Firouzeh; Pourbagher, Roghayeh


    Persian shallot, also called leek (Allium ampeloprasum), is a monocotyledon plant of the lily family (Liliaceae). It belongs to the genus Allium, has a characteristic taste and morphological features, making it to be considered as one of the popular herbal medicine. This research was conducted with the purpose of obtaining optimal conditions for tissue culture of Persian shallot and comparing its active ingredient production in vitro versus in vivo. In this study, the auxin 2, 4-D and benzyl aminopurine- 6 (BAP) hormones, each at two concentrations (0.5 and 0.1 mg/ L) and Kin at 0.5 mg/ L were used in the format of a randomized complete block design in three replications. Results showed that the best culture media for callus formation for leaf and seed explants were the MS cultures with the hormonal compositions (0.5 mg/ L of 2, 4- D, 0.1 mg/ L of BAP) and (0.5 mg/ L of Kin and 0.1 mg/ L of 2, 4- D). Identification of the chemical composition of the essential oils, extracted either from leek callus or leaf was carried out using GC mass analysis. Twenty one compounds were detected in the GC mass spectra, seven of which constitutv about 51.5% of the total amount of compounds present in the essential oils were identified. Our data demonstrate that the leek essential oil constituents as well as callus formation can be affected by culture medium condition.

  6. Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom (United States)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tracey L. (Inventor)


    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural, and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  7. Metabolic characterization of cultured mammalian cells by mass balance analysis, tracer labeling experiments and computer-aided simulations. (United States)

    Okahashi, Nobuyuki; Kohno, Susumu; Kitajima, Shunsuke; Matsuda, Fumio; Takahashi, Chiaki; Shimizu, Hiroshi


    Studying metabolic directions and flow rates in cultured mammalian cells can provide key information for understanding metabolic function in the fields of cancer research, drug discovery, stem cell biology, and antibody production. In this work, metabolic engineering methodologies including medium component analysis, (13)C-labeling experiments, and computer-aided simulation analysis were applied to characterize the metabolic phenotype of soft tissue sarcoma cells derived from p53-null mice. Cells were cultured in medium containing [1-(13)C] glutamine to assess the level of reductive glutamine metabolism via the reverse reaction of isocitrate dehydrogenase (IDH). The specific uptake and production rates of glucose, organic acids, and the 20 amino acids were determined by time-course analysis of cultured media. Gas chromatography-mass spectrometry analysis of the (13)C-labeling of citrate, succinate, fumarate, malate, and aspartate confirmed an isotopically steady state of the cultured cells. After removing the effect of naturally occurring isotopes, the direction of the IDH reaction was determined by computer-aided analysis. The results validated that metabolic engineering methodologies are applicable to soft tissue sarcoma cells derived from p53-null mice, and also demonstrated that reductive glutamine metabolism is active in p53-null soft tissue sarcoma cells under normoxia.

  8. Tissue characterization in cerebral ischemia using multiparameter MRI (United States)

    Soltanian-Zadeh, Hamid; Hammoud, Rabih; Jacobs, Michael A.; Patel, Suresh C.; Mitsias, Panayiotis D.; Pasnoor, Mamatha; Knight, Robert; Zheng, Zhang G.; Lu, Mei; Chopp, Michael


    After pre-processing and segmentation, the proposed method scores tissue regions between 1 and N. Score 1 is assigned to normal white matter and score N to CSF. Lesion zones are assigned a score based on their relative levels of similarities to white matter and CSF. To evaluate the method, 15 rats were imaged by a 7T MRI system at one of the three time points (acute, sub-acute, chronic) after MCA occlusion. Then, they were sacrificed and their brains were sliced and prepared for histological studies. MRI of 2 or 3 slices of each rat brain, using 2 DWI (b equals 400, b equals 800), 1 PDWI, 1 T2WI, and 1 T1WI, was used and an MRI score between 1 and 100 (N equals 100) was found for each region. Segmented regions were mapped onto the histology images and were scored by an experienced pathologist, from 1 to 10. MRI scores were validated using histology scores. To this end, correlation coefficients between the two scores (MRI and histology) were found. The results showed excellent correlations between MRI and histology scores at different time points.

  9. Micropatterned, clickable culture substrates enable in situ spatiotemporal control of human PSC-derived neural tissue morphology. (United States)

    Knight, G T; Sha, J; Ashton, R S


    We describe a modular culture platform that enables spatiotemporal control of the morphology of 2D neural tissues derived from human pluripotent stem cells (hPSCs) by simply adding clickable peptides to the media. It should be widely applicable for elucidating how spatiotemporal changes in morphology and substrate biochemistry regulate tissue morphogenesis.

  10. Breast tissue characterization using FARMA modeling of ultrasonic RF echo. (United States)

    Alacam, Burak; Yazici, Birsen; Bilgutay, Nihat; Forsberg, Flemming; Piccoli, Catherine


    A number of empirical and analytical studies demonstrated that the ultrasound RF echo reflected from tissue exhibits 1/f characteristics. In this paper, we propose to model 1/f characteristics of the ultrasonic RF echo by a novel parsimonious model, namely the fractional differencing auto regressive moving average (FARMA) process, and evaluated diagnostic value of model parameters for breast cancer malignancy differentiation. FARMA model captures the fractal and long term correlated nature of the backscattered speckle texture and facilitates robust efficient estimation of fractal parameters. In our study, in addition to the computer generated FARMA model parameters, we included patient age and radiologist's prebiopsy level of suspicion (LOS) as potential indicators of malignant and benign masses. We evaluated the performance of the proposed set of features using various classifiers and training methods using 120 in vivo breast images. Our study shows that the area under the receiver operating characteristics (ROC) curve of FARMA model parameters alone is superior to the area under the ROC curve of the radiologist's prebiopsy LOS. The area under the ROC curve of the three sets of features yields a value of 0.87, with a confidence interval of [0.85, 0.89], at a significance level of 0.05. Our results suggest that the proposed method of ultrasound RF echo model leads to parameters that can differentiate breast tumors with a relatively high precision. This set of RF echo features can be incorporated into a comprehensive computer-aided diagnostic system to aid physicians in breast cancer diagnosis.

  11. MRI characterization of brown adipose tissue in obese and normal-weight children

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Jie; Rigsby, Cynthia K.; Shore, Richard M. [Ann and Robert H. Lurie Children' s Hospital of Chicago, Department of Medical Imaging, 225 E. Chicago Ave., Box 9, Chicago, IL (United States); Northwestern University, Department of Radiology, Feinberg School of Medicine, Chicago, IL (United States); Schoeneman, Samantha E. [Ann and Robert H. Lurie Children' s Hospital of Chicago, Department of Medical Imaging, 225 E. Chicago Ave., Box 9, Chicago, IL (United States); Zhang, Huiyuan [John H. Stroger, Jr. Hospital of Cook County, Collaborative Research Unit, Chicago, IL (United States); Kwon, Soyang [Ann and Robert H. Lurie Children' s Hospital of Chicago, Stanley Manne Children' s Research Institute, Chicago, IL (United States); Northwestern University, Department of Pediatrics, Feinberg School of Medicine, Chicago, IL (United States); Josefson, Jami L. [Ann and Robert H. Lurie Children' s Hospital of Chicago, Division of Endocrinology, Chicago, IL (United States); Northwestern University, Department of Pediatrics, Feinberg School of Medicine, Chicago, IL (United States)


    Brown adipose tissue (BAT) is identified in mammals as an adaptive thermogenic organ for modulation of energy expenditure and heat generation. Human BAT may be primarily composed of brown-in-white (BRITE) adipocytes and stimulation of BRITE may serve as a potential target for obesity interventions. Current imaging studies of BAT detection and characterization have been mainly limited to PET/CT. MRI is an emerging application for BAT characterization in healthy children. To exploit Dixon and diffusion-weighted MRI methods to characterize cervical-supraclavicular BAT/BRITE properties in normal-weight and obese children while accounting for pubertal status. Twenty-eight healthy children (9-15 years old) with a normal or obese body mass index participated. MRI exams were performed to characterize supraclavicular adipose tissues by measuring tissue fat percentage, T2*, tissue water mobility, and microvasculature properties. We used multivariate linear regression models to compare tissue properties between normal-weight and obese groups while accounting for pubertal status. MRI measurements of BAT/BRITE tissues in obese children showed higher fat percentage (P < 0.0001), higher T2* (P < 0.0001), and lower diffusion coefficient (P = 0.015) compared with normal-weight children. Pubertal status was a significant covariate for the T2* measurement, with higher T2* (P = 0.0087) in pubertal children compared to prepubertal children. Perfusion measurements varied by pubertal status. Compared to normal-weight children, obese prepubertal children had lower perfusion fraction (P = 0.003) and pseudo-perfusion coefficient (P = 0.048); however, obese pubertal children had higher perfusion fraction (P = 0.02) and pseudo-perfusion coefficient (P = 0.028). This study utilized chemical-shift Dixon MRI and diffusion-weighted MRI methods to characterize supraclavicular BAT/BRITE tissue properties. The multi-parametric evaluation revealed evidence of morphological differences in brown

  12. Skeletal muscle regeneration via engineered tissue culture over electrospun nanofibrous chitosan/PVA scaffold. (United States)

    Kheradmandi, Mahsa; Vasheghani-Farahani, Ebrahim; Ghiaseddin, Ali; Ganji, Fariba


    Skeletal muscle tissue shows a remarkable potential in regeneration of injured tissue. However, in some of chronic and volumetric muscle damages, the native tissue is incapable to repair and remodeling the trauma. In the same condition, stem-cell therapy increased regeneration in situations of deficient muscle repair, but the major problem seems to be the lack of ability to attachment and survive of injected cells on the exact location. In this study, chitosan/poly(vinyl alcohol) nanofibrous scaffold was studied to promote cell attachment and provide mechanical support during regeneration. Scaffold was characterized using scanning electron microscope, X-ray diffraction, and tensile test. Degradation and swelling behavior of scaffold were studied for 20 days. The cell-scaffold interaction was characterized by MTT assay for 10 days and in vivo biocompatibility of scaffold in a rabbit model was evaluated. Results showed that cells had a good viability, adhesion, growth, and spread on the scaffold, which make this mat a desirable engineered muscular graft. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1720-1727, 2016.

  13. Application of plant cell and tissue culture for the production of phytochemicals in medicinal plants. (United States)

    Pant, Bijaya


    Approximately 80% of the world inhabitants depend on the medicinal plants in the form of traditional formulations for their primary health care system well as in the treatment of a number of diseases since the ancient time. Many commercially used drugs have come from the information of indigenous knowledge of plants and their folk uses. Linking of the indigenous knowledge of medicinal plants to modern research activities provides a new reliable approach, for the discovery of novel drugs much more effectively than with random collection. Increase in population and increasing demand of plant products along with illegal trade are causing depletion of medicinal plants and many are threatened in natural habitat. Plant tissue culture technique has proved potential alternative for the production of desirable bioactive components from plants, to produce the enough amounts of plant material that is needed and for the conservation of threatened species. Different plant tissue culture systems have been extensively studied to improve and enhance the production of plant chemicals in various medicinal plants.

  14. Experiment on Tissue Culture Technique of Saposhnikovia divaricata(Turcz.) Schischk

    Institute of Scientific and Technical Information of China (English)

    TONG Weishuang; FAN Ruifeng; GUO Shicheng; CHANG Ying


    The study aimed to optimize the induction and differentiation medium by exploreing different tissue culture of Saposhnikovia divaricata (Turcz.) Schischk. In tissue culture with the root, stem segments, young leaf, cotyledonary node and axillary bud of Saposhnikovia divaricata (Turcz.) Schischk as explants, a lot of plantleles were obtained and the corresponding plant regeneration-system was established. The results showed that when use MS+1.0 mg·L-1 6-BA +0.2 mg·L-1 NAA as callus induction medium, the cotyledonary node had the highest bourgeon rate, and its callus was better than any others; MS +2 mg·L-1 6-BA +0.4 mg·L-1NAA was the best adventitious buds induction medium, and the best adventitious buds induced condition was 3% sucrose as carbon source, illumination for 12-14 h·d-1 and pH 5.8. The best rootage medium was 1/2 MS+0.5 mg·L-1 NAA.

  15. Screenhouse and field persistence of nonpathogenic endophytic Fusarium oxysporum in Musa tissue culture plants. (United States)

    Paparu, Pamela; Dubois, Thomas; Gold, Clifford S; Niere, Björn; Adipala, Ekwamu; Coyne, Daniel


    Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4-16 weeks after inoculation), root colonization did not. However, in the field (17-33 weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.

  16. Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications


    Costa, Pedro Ferreira da


    Programa Doutoral em Engenharia Biomédica In a typical tissue engineering approach, cells are collected from the patient and then seeded into a threedimensional scaffold where they proliferate to generate a tissue-like substitute to be re-implanted back into the defect site. However, human tissues possess various degrees of complexity which often makes them impossible to be reproduced in such a simplified way. In fact, many tissues such as bone, for example, exhibit specific ar...

  17. Radioimmunoassay and characterization of enkephalins in rat tissues

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.J.; Chang, K.J.; Cooper, B.; Cuatrecasas, P.


    A highly sensitive and specific radioimmunoassay for three enkephalins (opiate-like pentapeptides) has been developed. The assay utilizes /sup 125/I-labeled enkephalins and antisera raised in rabbits, to synthetic enkephalins coupled with glutaraldehyde to bovine serum albumin. These antisera show <1% cross-reactivity between H/sub 2/N-Tyr-Gly-Gly-Phe-Met-OH ((Met/sup 5/)enkephalin) and H/sub 2/N-Tyr-Gly-Gly-Phe-Leu-OH ((Leu/sup 5/)enkephalin) and even lower cross-reactvity to ..beta..-endorphin. Morphine shows no cross reactivity at all. The assay can detect as little as 10 fmol of enkephalin. Dose response curves for synthetic enkephalin and enkephalin-like immunoreactivity in acid extracts of brain are superimposable. Enkephalin-like immunoreactivity in tissue extracts is destroyed by treating extracts with leucine aminopeptidase or carboxypeptidase, enzymes which also destroy synthetic enkephalin. An opiate receptor binding assay based on the binding of /sup 125/I-labeled (DAla/sup 2/Leu/sup 5/)-enkephalin to N4TG1 neuroblastoma cells has also been developed. Gel filtration of acid extracts of brain or pituitary gland shows that both contain opiate-like material. However, whereas in brain most of opiate-like material co-chromatographs with enkephalin, in the pituitary no opiate-like material was observed to co-chromatograph with enkephalin but was of higher molecular weight. Enkephalin is widely distributed in the brain. High concentrations of both (Met/sup 5/)- and (Leu/sup 5/)enkephalins are found in the striatum (approximately 5 pmol/mg of protein), lower concentrations in the thalamus and midbrain, and very low concentrations in the cerebellum. The ratio of (Met/sup 5/)enkephalin to (Leu/sup 5/)enkephalin also differs in different brain areas. The ratio is very high in the hippocampus (15.2) and hypothalamus (13.6), but lower in other areas such as the cortex (1.4).

  18. Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants. (United States)

    Smulders, M J; Rus-Kortekaas, W; Vosman, B


    The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.

  19. Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture

    Institute of Scientific and Technical Information of China (English)

    Ya Huei CHEN; Yi Jung TSAI; Jian Zhi HUANG; Fure Chyi CHEN


    Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism,signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and posttranscriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed. ⅲ€Keywords: Phalaenopsis, tissue culture, peloric mutant, cDNA-RAPD, suppression subtractive hybridization.

  20. Organoid Culture of Isolated Cells from Patient-derived Tissues with Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Bing-Ying Xie; Ai-Wen Wu


    Background:Colorectal cancer (CRC) is a heterogeneous disease;current research relies on cancer cell lines and animal cancer models,which may not precisely imitate inner human tumors and guide clinical medicine.The purpose of our study was to explore and further improve the process of producing three-dimensional (3D) organoid model and impel the development of personalized therapy.Methods:We subcutaneously injected surgically resected CRC tissues from a patient into BALB/c-nu mice to build patient-derived xenografts (PDXs).Isolated cells from PDXs at appropriate tumor size were mingled with Matrigel,and then seeded in ultra-low attachment 96-well plates at four cell densities (500,1000,2000,and 4000 single cells/well).Cells were cultured with advanced Dulbecco's Modified Eagle Mediurn/F 12 medium additional with various factors added to maintain tumor's biological traits and growth activity.The growth curves of the four cell densities were measured after 24 h of culture until 25 days.We evaluated the effects of four chemotherapeutic agents on organoid model by the CellTiter-Glo(R) Luminescent Cell Viability Assay.Hematoxylin and eosin (H and E) staining of 3D organoids was performed and compared with patient and CRC PDX tissues.Furthermore,immunohistochemistry was performed,in which the organoids were stained with the proliferation marker,Ki-67.During the experimental process,a phase-contrast microscope was used.Results:Phenotype experimental results showed that 3D organoids were tightly packed together and grew robustly over time.All four densities of cells formed organoids while that composed of 2000 cells/well provided an adequate cultivation system and grew approximately 8-fold at the 25th day.The chemosensitivity of the four conventional drugs was [s]-l 0-hydroxycamptothecin > mitomycin C > adriamycin > paclitaxel,which can guide clinical treatment.Histological features of CRC patient's tumor tissues and mice tumor xenograft tissues were highly similar

  1. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies. (United States)

    Duressa, Tewodros Firdissa; Huybrechts, Roger


    Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.


    Directory of Open Access Journals (Sweden)

    V. V. Ivanov


    Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone ahillin in hepatoma tissue culture (HTC cells.Material and methods. In this study we’ve evaluated the effect of γ-lactone sesquiterpene aсhillin and gemfibrozil (comparator drug on the lipid content in the hepatoma tissue culture (HTC cell which were incubated with a fat emulsion lipofundin by fluorescent method with vital dye Nile Redand staining the cells with the dye Oil Red O. The cell viability was investigated using the MTT-test and staining with Trypan blue.Results. Cultivation cells HTC with aсhillin and gemfibrozilat concentrations ranging from 0.5 to1.5 mM and from0.25 mM to0.5 mM, respectively, resulted in dose-dependent decrease of the fluorescence’s intensity Nile Red. It reflects a decrease in lipid content in the cells. At these concentrations the drugs didn’t have cytotoxic effect and the cell viability didn’t change compared to the control culture.An experimental hyperlipidemia in the hepatoma culture cells was induced by adding to the incubation medium a fat emulsion lipofundin at a final concentration 0.05%. The intensity of fluorescence Nile Red in the cells was increased 4 fold (p < 0.05. This result suggests the significant accumulation of lipids in the cell’s cytosol and confirmed by microscopy after staining neutral lipids with the dye Oil Red O. Under these conditions aсhillin and gemfibrozil reduced lipid content in cells and hadthe effect at concentrations of0.5 mM and0.25 mM respectively.Conclusion. In the lipofundin-mediated model of hyperlipidemia the sesquiterpene lactone aсhillin prevents the lipid accumulation in cells. It confirms by decrease of fluorescence Nile Red and reduction lipid drops which were stained with Oil Red O in cytosol. To establish the molecular targets of aсhillin’saction on lipid metabolism in cell culture HTC we need to investigate a gene expression of key enzymes of lipid metabolism.

  3. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Chunlei Miao


    Full Text Available Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious.

  4. Osteogenic Differentiation Capacity of In Vitro Cultured Human Skeletal Muscle for Expedited Bone Tissue Engineering (United States)

    Miao, Chunlei; Zhou, Lulu; Tian, Lufeng; Zhang, Yingjie; Zhang, Wei; Yang, Fanghong; Liu, Tianyi


    Expedited bone tissue engineering employs the biological stimuli to harness the intrinsic regenerative potential of skeletal muscle to trigger the reparative process in situ to improve or replace biological functions. When genetically modified with adenovirus mediated BMP2 gene transfer, muscle biopsies from animals have demonstrated success in regenerating bone within rat bony defects. However, it is uncertain whether the human adult skeletal muscle displays an osteogenic potential in vitro when a suitable biological trigger is applied. In present study, human skeletal muscle cultured in a standard osteogenic medium supplemented with dexamethasone demonstrated significant increase in alkaline phosphatase activity approximately 24-fold over control at 2-week time point. More interestingly, measurement of mRNA levels revealed the dramatic results for osteoblast transcripts of alkaline phosphatase, bone sialoproteins, transcription factor CBFA1, collagen type I, and osteocalcin. Calcified mineral deposits were demonstrated on superficial layers of muscle discs after an extended 8-week osteogenic induction. Taken together, these are the first data supporting human skeletal muscle tissue as a promising potential target for expedited bone regeneration, which of the technologies is a valuable method for tissue repair, being not only effective but also inexpensive and clinically expeditious. PMID:28210626

  5. Primary Establishment of the Tissue Culture Technique and Regeneration System for Ornamental Lupinns polyphyllus

    Institute of Scientific and Technical Information of China (English)

    WANG Xiaoling; GAO Zhu; LIU Tengyun; YU Faxin; WANG Biqin


    The purpose of this paper is to develop a system for tissue culture and rapid propagation of two ornamental lupins, Minaretie and Russell Prize. In view of screening out the better explant regeneration and suitable culture medium, through adding hormone 6-B A, NAA and 2,4-D into MS and B5 basic culture medium, a series of experiments were carried out with the shoot tips, leaves, leaf petioles and stems from the asepsis seedling. The results showed that the shoot tips had favorableness on the rapidly propagation; MS+6-BA 0.5 rng·L-1 for first generation, the induction rate of Minaretie and Russell Prize was 90.5% and 95.86% respectivdy; Minaretie had the highest propagation index (6.35) on MS+6-BA 0.5 mg·L-1+NAA 0 mg·L-1+GA 30.8 mg·L-1+AC 2 g·L-1, but Russell Prize had the highest propagation index (7.24) on MS+6-BA 0.5 mg·L-1+NAA0.15 mg ·L-1+GA3 1.0 mg·L-1+AC 0.5 g·L-1; 1/2 MS+NAA 0.25 mg·L-1 was the best rooting medium. The ratios of getting roots of Minaretie and Russell Prize were 94.78% and 96.32%, respectively.

  6. 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.

  7. Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps). (United States)

    Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E


    Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.

  8. Potential for tissue characterization of plaque and arterial wall using intravascular ultrasound (United States)

    Sudhir, Krishnakutty; Fitzgerald, Peter J.; Yock, Paul G.


    Current intravascular ultrasound imaging technology is able to determine the extent and distribution of pathologic processes within the vessel wall, but is not highly sensitive in discriminating between different types of pathologic tissue. `Tissue characterization' refers to a set of computer-based techniques that utilize features of the ultrasound signal beyond basic amplitude to help define the structure of the tissue of interest. Although preliminary results with this approach are encouraging, additional work is needed to define its clinical application in vascular disease.

  9. Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.

    Directory of Open Access Journals (Sweden)

    Satoru Moritoh

    Full Text Available BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL per retina, a higher speed (constant 55 rpm of agitation by rotary shaker, and a greater concentration (10% of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age. The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing

  10. Measurement of Cu/Zn SOD in placenta, cultured cells, various fetal tissues, decidua and semen by ELISA. (United States)

    Ali Akbar, S; Nicolaides, K H; Brown, P R


    The concentration of copper/zinc-containing superoxide dismutase (Cu/Zn SOD) was measured in placental villous tissues (8-20 weeks' gestation), decidual tissues, cultured cells from chorionic villi and amniotic fluid cells, various fetal tissues (8-11 weeks' gestation), spermatozoa, seminal plasma and ovarian follicular fluid using a sensitive enzyme-linked immunosorbent assay (ELISA). The isoenzyme was expressed in all samples expect ovarian follicular fluid. Cu/Zn SOD was also detected in hydatidiform mole and choriocarcinoma. In placental villous tissues the concentration of the enzyme increased with gestation between 8 and 20 weeks of pregnancy (n = 69, r = 0.34, P < 0.005).

  11. Transgenic Crops by Direct Treatment of Exogenous DNA Without Agrobacterium tumefaciens Plasmid and Tissue Culture

    Institute of Scientific and Technical Information of China (English)



    Gene transfter methods are developing quickly recently,but each method has its limitations.We introduce a new gene transfer technique in this paper,which is simple,effective,and easy to operate,but does not get enough attention from scientists.This technique is used to transform plants by injecting exogenous DNA to stigma,style,ovary,young fruit or meristem of the recipient,or soaking the recipient's seeds in exogenous DNA solution.Los of heritable variations were found in many characters of many crops,It may be used to creaste new germplasms or realize gene exchange between different species,gerera,or families,even between animals and plants,A brief discussion was given to the mechanism of exogenous DNA introduction,integration into and expression in the recipient.We also discussed the merits and limitations of the technique.Currently there are two successful approaches that can be used to transform plants genetically,but each method has its limitations that are delaying the application of the techniques to certaincommercially important crops.The first tecnhique exploits a natural genetic engineer,Agrobacterium tumefaciens,which contains a tumor-inducing(Ti) plasmid that transfers a DNA segment(the T-DNA) from the plasmid to the nuclear genome of infected plants(or in vitro to plant tissue).The method is restricted to dicotyledenous plants;monocotyledenous plants are usually not susceptible to agrobacterial infection.The second technique involves direct transfter of DNA to plant protoplast ,prepared by enzymatic digestion of cell walls,for example by chemically stimulated uptake using polyethylene glycol or a high voltage pulse,generating transient'holes'in the protoplast membrane.This technique depends on a tissue culture system that allows regeneration of mature plants from protoplasts,But so far it is impossible to achieve plant regeneration from protoplasts in many crops.Both techniques use dominant selectable markers(for example,kanamycin resistance) to

  12. 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.

  13. Potential application of urea-derived herbicides as cytokinins in plant tissue culture. (United States)

    Srinivasan, Malathi; Nachiappan, Vasanthi; Rajasekharan, Ram


    Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%)with a mean number of 5 shoots in alfalfa, in C.forskohlii, the frequency of regeneration was 90%with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

  14. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik


    In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without...... a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole...... and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption...

  15. Advanced tissue culture used by Twyfords to build up jojoba clones

    Energy Technology Data Exchange (ETDEWEB)


    Twyford Plant Laboratories Ltd. in the UK, using their own advanced methods of plant tissue culture, have built up a bank of 30 different male and female clones of jojoba, the arid land crop whose seeds produced a liquid wax which - amongst other uses - can be substituted for sperm whale oil. The technique involves growing microscopic parts of a parent plant on a medium containing all the necessary growth hormones, salts, vitamins and other nutrients. Growth takes place under artificial light in an all-electric controlled, air-conditioned environment. No other method is so successful for rapidly multiplying plants, particularly those that do not breed true from seed. These include most fruits and some flowers and vegetables.

  16. Potential application of urea-derived herbicides as cytokinins in plant tissue culture

    Indian Academy of Sciences (India)

    Malathi Srinivasan; Vasanthi Nachiappan; Ram Rajasekharan


    Various urea-derived herbicides and different cytokinin analogues were used to determine their effects on callusing response and shoot regenerating capacity of alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides monuron and diuron evoked profuse callusing response from Coleus leaf segments and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l) showed a high frequency of shoot regeneration (89%) with a mean number of 5 shoots in alfalfa, in C. forskohlii, the frequency of regeneration was 90% with a mean number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed significantly higher cytokinin-like activity than single chloride substitution monuron. This study demonstrates the potential use of monuron and diuron as cytokinins in plant tissue culture.

  17. 细辛组织培养研究%Tissue Culture of Asarum siboldii

    Institute of Scientific and Technical Information of China (English)

    南桂仙; 金光德


    以细辛叶片为外植体,研究了其组织培养技术.结果表明:愈伤组织继代1次,可增殖10~15倍,每块愈伤组织经过再分化能长出5~8株试管苗,反复继代3~4次,每个叶片可产出约500株试管苗.%Used the leaf of Asarum siboldii as explants,the tissue culture technique was studied.The results showed that the the callus could proliferate 10-15 times after subculturing for one time.5-8 plantlets grew out when the callus dedifferentiated.When the callus was subcultured for 3-4 times,about 500 plantlets could be obtained.

  18. DNA polymorphism in Cab locus of tomato induced by tissue culture. (United States)

    Nambisan, P; Chopra, V L; Mohapatra, T


    Plants were regenerated from callus induced from leaf disc explants of a tomato F1 hybrid heterozygous for three marker loci anthocyaninless (a), without anthocyanin (aw), and hairless (hl). Regenerants were studied for somaclonal variation at the phenotypic level by scoring for variation in the marker loci, and at the DNA level by probing geomic DNA blots with a chlorophyll a/b binding protein (Cab-3C) cDNA sequence. While no variation was observed at the phenotypic level in over 950 somaclones studied, DNA polymorphism for the Cab locus could be detected in two out of 17 somaclones tested. Tissue culture induced variation at the phenotypic level for specific loci is very low (less than 0.001 for a, aw or hl) but DNA sequence changes are induced at much greater frequency (approximately 0.1 for a multicopy gene family such as Cab).

  19. Plant regeneration from petiole segments of some species in tissue culture

    Directory of Open Access Journals (Sweden)

    Krystyna Klimaszewska


    Full Text Available The regeneration ability of 21 plant species belonging to 14 families was tested. The method of tissue culture in vitro was applied, on basic MS medium with an addition of growth regulators from the auxin and cytokinin groups. From among the investigated plant groups Peperomia scandens and Caladium × hortulanum were capable of plant regeneration, Passiilora coerulea regenerated shoots, Hedera helix, Begonia glabra, Coleus blumei, Fuchsia hybrida, Passiflora suberosa and Peperomia eburnea formed callus and roots, Kalanchoe blossfeldiana, Pelargonium grandiflorum, P. peltatum, P. radula, Coleus shirensis and Magnolia soulangeana produced callus, Philodendron scandens, Rhododendron smirnovii, Hibiscus rosa-sinensis, Coprosma baueri, Cestrum purpureum and Solanum rantonnetii did not exhibit any regeneration reactions.

  20. Cloning crops in a CELSS via tissue culture: Prospects and problems (United States)

    Carman, John G.; Hess, J. Richard


    Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries.

  1. Tissue culture of adult larch as a tool for breeding purposes

    Energy Technology Data Exchange (ETDEWEB)

    Ewald, D.; Kretzschmar, U. [Federal Research Centre of Forestry and Forest Products, Waldsieversdorf (Germany). Inst. for Forest Tree Biology


    Aimed at the identical reproduction of genotypes which are considered superior different methods were tested to establish and to propagate tissue cultures from old larch trees (L. decidua, L. kaempferi, L. sukaczewii, L. gmelinii, L. eurolepis). Serial subcultures without phytohormones (shoot tip propagation) led to the establishment of clone lines. After ten subcultures propagation velocity, shoot morphology and rooting behavior were similar to juvenile plant material. Serial subcultures which included a cytokinin induction led to the formation of adventitious shoot clusters (adventitious bud propagation). Adventitious shoots derived from male flowers of one L. kaempferi clone could be propagated via shoot tip propagation. Micrografting of meristems in vitro resulted in a regained rooting capacity of green cuttings from micrografts. Combining these in vitro techniques offers now the possibility to propagate selected mature larch trees for different breeding purposes. 23 refs, 5 figs, 2 tabs

  2. Preparation and characterization of gelatin–hydroxyapatite composite microspheres for hard tissue repair

    Energy Technology Data Exchange (ETDEWEB)

    Chao, Shao Ching [Department of Materials Science and Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Department of Minimally Invasive Skull Neurosurgery, Neurological Institute, Taichung Veterans General Hospital, 1650 Taiwan Boulevard, Sect. 4, Taichung, Taiwan (China); Department of Neurosurgery, ChangHua Hospital, Ministry of Health and Welfare, 80 Chung Cheng Road, Sect. 2 Chiu Kuan Village, Changhua 500, Taiwan (China); Wang, Ming-Jia; Pai, Nai-Su [Department of Materials Science and Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China); Yen, Shiow-Kang, E-mail: [Department of Materials Science and Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (China)


    Gelatin–hydroxyapatite composite microspheres composed of 21% gelatin (G) and 79% hydroxyapatite (HA) with uniform morphology and controllable size were synthesized from a mixed solution of Ca(NO{sub 3}){sub 2}, NH{sub 4}H{sub 2}PO{sub 4} and gelatin by a wet-chemical method. Material analyses such as X-ray diffraction (XRD), scanning/transmission electron microscopy examination (SEM/TEM) and inductively coupled plasma-mass spectroscopy (ICP-MS) were used to characterize G–HA microspheres by analyzing their crystalline phase, microstructure, morphology and composition. HA crystals precipitate along G fibers to form nano-rods with diameters of 6–10 nm and tangle into porous microspheres after blending. The cell culture indicates that G–HA composite microspheres without any toxicity could enhance the proliferation and differentiation of osteoblast-like cells. In a rat calvarial defect model, G–HA bioactive scaffolds were compared with fibrin glue (F) and Osteoset® Bone Graft Substitute (OS) for their capacity of regenerating bone. Four weeks post-implantation, new bone, mineralization, and expanded blood vessel area were found in G–HA scaffolds, indicating greater osteoconductivity and bioactivity than F and OS. - Highlights: • G–HA composite microspheres were prepared by hydroxyapatite and gelatin. • In vitro tests indicated that the G–HA microspheres were biocompatible and bioactive. • In in vitro tests, G–HA microspheres could be applied in hard tissue engineering. • G–HA had healed the bone defect and provides a high proportion of surface area to open space.

  3. Isolation, Characterization and Virulence Potential of Tenacibaculum dicentrarchi in Salmonid Cultures in Chile. (United States)

    Avendaño-Herrera, R; Irgang, R; Sandoval, C; Moreno-Lira, P; Houel, A; Duchaud, E; Poblete-Morales, M; Nicolas, P; Ilardi, P


    In this study, we isolated, identified and characterized isolates of Tenacibaculum dicentrarchi in Atlantic salmon (Salmo salar) farmed in Chile for the first time. In 2010 and 2014, mortalities were observed in Atlantic salmon (average weight 25-30 and 480-520 g, respectively) at an aquaculture centre in Puerto Montt, Chile. Severe tail rots, frayed fins and, in some cases, damaged gills were detected. Wet smear analyses of these lesions revealed a high occurrence of Gram-negative, filamentous bacteria. Microbiological analysis of infected gill and tail tissues yielded six bacterial isolates. All were identified as T. dicentrarchi through polyphasic taxonomy, which included phenotypic characterization, 16S rRNA sequencing and multilocus sequence typing. The latter method revealed a close relationship of the Chilean genotype with the T. dicentrarchi type strain and two Norwegian Atlantic cod (Gadus morhua) isolates. The pathogenic potential of the TdChD05 isolate was assessed by challenging Atlantic salmon and rainbow trout (Oncorhynchus mykiss) for one hour, which resulted in mean cumulative mortality rates of 65% and 93%, respectively, as well as clinical signs 14 days post-challenge. However, challenged Coho salmon (Oncorhynchus kisutch) presented no mortalities or clinical signs of infection. These findings indicate that the geographical and host distribution of T. dicentrarchi is wider than previously established and that this bacterium may have negative impacts on salmonid cultures.

  4. Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes. (United States)

    Katsura, K; Mellergård, P; Theander, S; Ouyang, Y B; Siesjö, B K


    The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

  5. Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue.

    Directory of Open Access Journals (Sweden)

    Darryl R Davis

    Full Text Available BACKGROUND: At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs. Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential of CDCs. METHODOLOGY/PRINCIPAL FINDINGS: CPCs were expanded from human endomyocardial biopsies (n = 160, adult bi-transgenic MerCreMer-Z/EG mice (n = 6, adult C57BL/6 mice (n = 18, adult GFP(+ C57BL/6 transgenic mice (n = 3, Yucatan mini pigs (n = 67, adult SCID beige mice (n = 8, and adult Wistar-Kyoto rats (n = 80. Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit(+, endothelial cells (CD31(+, CD34(+, and mesenchymal cells (CD90(+. Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that

  6. Synthesis, characterization and antioxidant activity of a novel electroactive and biodegradable polyurethane for cardiac tissue engineering application. (United States)

    Baheiraei, Nafiseh; Yeganeh, Hamid; Ai, Jafar; Gharibi, Reza; Azami, Mahmoud; Faghihi, Faezeh


    There has been a growing trend towards applying conducting polymers for electrically excitable cells to increase electrical signal propagation within the cell-loaded substrates. A novel biodegradable electroactive polyurethane containing aniline pentamer (AP-PU) was synthesized and fully characterized by spectroscopic methods. To tune the physico-chemical properties and biocompatibility, the AP-PU was blended with polycaprolactone (PCL). The presence of electroactive moieties and the electroactivity behavior of the prepared films were confirmed by UV-visible spectroscopy and cyclic voltammetry. A conventional four probe analysis demonstrated the electrical conductivity of the films in the semiconductor range (~10(-5)S/cm). MTT assays using L929 mouse fibroblast and human umbilical vein endothelial cells (HUVECs) showed that the prepared blend (PB) displayed more cytocompatibility compared with AP-PU due to the introduction of a biocompatible PCL moiety. The in vitro cell culture also confirmed that PB was as supportive as tissue culture plate. The antioxidant activity of the AP-PU was proved using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay by employing UV-vis spectroscopy. In vitro degradation tests conducted in phosphate-buffered saline, pH7.4 and pH5.5, proved that the films were also biodegradable. The results of this study have highlighted the potential application of this bioelectroactive polyurethane as a platform substrate to study the effect of electrical signals on cell activities and to direct desirable cell function for tissue engineering applications.

  7. Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

    Directory of Open Access Journals (Sweden)

    Gesiane Ribeiro


    Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

  8. A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering. (United States)

    Zhang, Zhi-Yong; Teoh, Swee Hin; Teo, Erin Yiling; Khoon Chong, Mark Seow; Shin, Chong Woon; Tien, Foo Toon; Choolani, Mahesh A; Chan, Jerry K Y


    Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

  9. Plant tissue culture of fast-growing trees for phytoremediation research. (United States)

    Couselo, José Luis; Corredoira, Elena; Vieitez, Ana M; Ballester, Antonio


    The ability of plants to remove pollutants from the environment is currently used in a simple and low-cost cleaning technology known as phytoremediation. Unfortunately, little is known about the metabolic pathways involved in the transformation of xenobiotic compounds and the ability of certain plants to tolerate, detoxify, and store high concentrations of heavy metals. Plant cell and tissue culture is considered an important tool for fundamental studies that provide information about the plant-contaminant relationships, help to predict plant responses to environmental contaminants, and improve the design of plants with enhanced characteristics for phytoremediation. Callus, cell suspensions, hairy roots, and shoot multiplication cultures are used to study the interactions between plants and pollutants under aseptic conditions. Many plant species have an inherent ability to accumulate/metabolize a variety of pollutants, but they normally produce little biomass. However, fast-growing trees are excellent candidates for phytoremediation because of their rapid growth, extensive root system, and high water uptake. This chapter outlines the in vitro plant production of both somaclonal variants and transgenic plants of Populus spp. that exhibit high tolerance to heavy metals.

  10. Effect of radiation-degraded chitosan on growth promotion of flower plant in tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Luan, Le Quang; Ha, Vo Thi Thu; Hai, Le; Hien, Nguyen Quoc [Vietnam Atomic Energy Commission, Nuclear Research Institute, Dalat (Viet Nam); Nagasawa, Naotsugu; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment


    Radiation is a useful tool for degradation of polysaccharides, such as starch, carrageenan, alginate and chitin/chitosan. The viscosity molecular weight (Mw) of chitosan with 80% degree of deacetylation was reduced to 1.5 x 10{sup 5} by irradiation of 50kGy in solid phase. The solution of 10% of chitosan with Mw ca. 15 x 10{sup 5} was then irradiated at doses ranging 10-250kGy for further degradation and the products were supplemented into cultural media for testing of plant growth promotion effect. The results indicated that irradiated chitosan showed a strong growth-promotion effect on the increase of the length of shoot, the length of root and fresh biomass for flower plants namely Limonium latifolium, Eustoma grandiflorum and Chrysanthemum morifolium in tissue culture. The growth-promotion effect was obtained by the treatments with 50ppm of chitosan irradiated at the doses of 75-100kGy in 10% solution. The suitable concentrations of chitosan irradiated at 100kGy are ca. 100ppm for C. morifolium, 30ppm for E. grandiflorum and 40ppm for L. latifolium. In addition, our study also indicated that the survival ratio of transferred flower plantlets treated with irradiated chitosan was improved after acclimatizing for 30 days in the greenhouse. Accordingly, it is concluded that degraded chitosan obtained by radiation degradation technique is effective as a plant growth promoter as well as irradiated alginate. (author)

  11. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Samuel D Robinson


    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

  12. Clonal multiplication of Cymbidiums through tissue culture of the shoot meristem

    Energy Technology Data Exchange (ETDEWEB)

    Wimber, Donald E.


    The propagation of clonal varieties of some orchids is at times exasperatingly slow and occasionally an almost futile effort. Clonal multiplication is generally confined to dlvidlng mature plants and to starting plants from pseudobulbs. There is, of course, the specialized technique for obtaining Phalaenopsis plantlets from the aseptic culture of inflorescence nodes, but this is basically the same thing as propagating plants from pseudobulbs. In certain cases it is highly desirable to rapidly multiply certain clones of orchids. Awarded varieties could thereby be dispersed with great rapidity where now it may take decades for some clones to became fairly common. Commercial flower production would be very much enhanced if certain desirable clones could be multiplied ad infinitum within a short time. Orchid flower production could then be placed more on a par with many of the other cut flowers and the clonal peculiarities of some fo the current hybrids could be pampered instead of ignored. This paper describes a tissue culture method for the rapid propagation of Cymbidium clones.

  13. Constructing Failure: Leonard Hayflick, Biomedicine, and the Problems with Tissue Culture. (United States)

    Park, Hyung Wook


    By examining the use of tissue culture in post-war American biomedicine, this paper investigates how scientists experience and manage failure. I study how Leonard Hayflick forged his new definition of failure and ways of managing it by refuting Alexis Carrel's definition of failure alongside his theory of the immortality of cultured cells. Unlike Carrel, Hayflick claimed that every vertebrate somatic cell should eventually die, unless it transformed into a tumour cell. This claim defined cell death, which had been a problem leading to a laboratory failure, as a normal phenomenon. On the other hand, permanent life, which had been considered a normal cellular characteristic, became a major factor causing scientific failure, since it implied malignant transformation that scientists hoped to control. Hayflick then asserted that his cell strains and method would partly enable scientists to manage this factor-especially that occurred through viral infection-alongside other causes of failure in routine tasks, including bacterial contamination. I argue that the growing biomedical enterprise fostered this work of Hayflick's, which had repercussions in both his career and the uses of cells in diverse investigations. His redefinition of failure in the age of biomedicine resulted in the broad dissemination of his cells, medium, and method as well as his long struggle with the National Institutes of Health (NIH), which caused his temporarily failed career.

  14. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas;


    -mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo......Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...

  15. Tissue Equivalent Phantom Design for Characterization of a Coherent Scatter X-ray Imaging System (United States)

    Albanese, Kathryn Elizabeth

    testing. Our imaging system has been able to define the location and composition of the various materials in the phantom. These phantoms were used to characterize the CACSSI system in terms of beam width and imaging technique. The result of this work showed accurate modeling and characterization of the phantoms through comparison of the tissue-equivalent form factors to those from literature. The physical construction of the phantoms, based on actual patient anatomy, was validated using mammography and computed tomography to visually compare the clinical images to those of actual patient anatomy.

  16. Comparative Study on Antioxidative System in Normal and Vitrified Shoots of Populus suaveolens in Tissue Culture

    Institute of Scientific and Technical Information of China (English)

    Lin Shanzhi; Zhang Zhiyi; Lin Yuanzhen; Liu Wenfeng; Guo Huan; Zhang Wei; Zhang Chong


    To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H2O2, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P. Suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period. While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H2O2, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H2O2. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the

  17. Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues. (United States)

    Fortier, Guillaume Marceau; Gauvin, Robert; Proulx, Maryse; Vallée, Maud; Fradette, Julie


    Mesenchymal cells are central to connective tissue homeostasis and are widely used for tissue-engineering applications. Dermal fibroblasts and adipose-derived stromal cells (ASCs) allow successful tissue reconstruction by the self-assembly approach of tissue engineering. This method leads to the production of multilayered tissues, devoid of exogenous biomaterials, that can be used as stromal compartments for skin or vesical reconstruction. These tissues are formed by combining cell sheets, generated through cell stimulation with ascorbic acid, which favours the cell-derived production/organization of matrix components. Since media motion can impact on cell behaviour, we investigated the effect of dynamic culture on mesenchymal cells during tissue reconstruction, using the self-assembly method. Tissues produced using ASCs in the presence of a wave-like movement were nearly twice thicker than under standard conditions, while no difference was observed for tissues produced from dermal fibroblasts. The increased matrix deposition was not correlated with an increased proliferation of ASCs, or by higher transcript levels of fibronectin or collagens I and III. A 30% increase of type V collagen mRNA was observed. Interestingly, tissues engineered from dermal fibroblasts featured a four-fold higher level of MMP-1 transcripts under dynamic conditions. Mechanical properties were similar for tissues reconstructed using dynamic or static conditions. Finally, cell sheets produced using ASCs under dynamic conditions could readily be manipulated, resulting in a 2 week reduction of the production time (from 5 to 3 weeks). Our results describe a distinctive property of ASCs' response to media motion, indicating that their culture under dynamic conditions leads to optimized tissue engineering.

  18. Cultural routes: characterization and challenges of a new category of world cultural heritage


    Martínez Yáñez, Celia; Universidad de Granada


    This paper studies in depth the progress that cultural routes, as a new category of World Cultural Heritage, introduce in heritageprotection, as a result of their multidimensional, transnational and wide territorial extension. For this purpose it focuses onboth its scientific definition and its differentiation from other heritage categories, as well as on the challenges that set out itsfull assumption in the framework of the Convention Concerning the Protection of World Cultural and Natural H...

  19. Characterization of thermoplastic polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by microcellular injection molding. (United States)

    Mi, Hao-Yang; Salick, Max R; Jing, Xin; Jacques, Brianna R; Crone, Wendy C; Peng, Xiang-Fang; Turng, Lih-Sheng


    Polylactic acid (PLA) and thermoplastic polyurethane (TPU) are two kinds of biocompatible and biodegradable polymers that can be used in biomedical applications. PLA has rigid mechanical properties while TPU possesses flexible mechanical properties. Blended TPU/PLA tissue engineering scaffolds at different ratios for tunable properties were fabricated via twin screw extrusion and microcellular injection molding techniques for the first time. Multiple test methods were used to characterize these materials. Fourier transform infrared spectroscopy (FTIR) confirmed the existence of the two components in the blends; differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) confirmed the immiscibility between the TPU and PLA. Scanning electron microscopy (SEM) images verified that, at the composition ratios studied, PLA was dispersed as spheres or islands inside the TPU matrix and that this phase morphology further influenced the scaffold's microstructure and surface roughness. The blends exhibited a large range of mechanical properties that covered several human tissue requirements. 3T3 fibroblast cell culture showed that the scaffolds supported cell proliferation and migration properly. Most importantly, this study demonstrated the feasibility of mass producing biocompatible PLA/TPU scaffolds with tunable microstructures, surface roughnesses, and mechanical properties that have the potential to be used as artificial scaffolds in multiple tissue engineering applications.

  20. A method to obtain reference images for evaluation of ultrasonic tissue characterization techniques

    DEFF Research Database (Denmark)

    Jensen, M.S.; Wilhjelm, Jens E.; Sahl, B.


    A general problem when evaluating ultrasonic methods for tissue characterization is that "a golden standard" is seldom known. This paper describes a manual method to obtain a reference image, with the same geometry as the ultrasound image, indicating spatial location of the different tissue types...... compound ultrasound images of tissue and fiducial markers were recorded each 0.5 mm. Guided by the fiducial markers, the agar block was subsequently cut into slices 2.5 mm thick, photographed and finally analyzed histologically identifying these tissues: collagen rich, collagen poor, micro vessels...... and muscle fibres. Due to: (1) the cutting procedure, (2) the finite size of the ultrasound beam and (3) the spatial variation in propagation velocity, the macroscopic photographs did not align completely with the ultrasound images. Likewise, the histological image is a geometrically distorted version...

  1. Tissue characterization using electrical impedance spectroscopy data: a linear algebra approach. (United States)

    Laufer, Shlomi; Solomon, Stephen B; Rubinsky, Boris


    In this study, we use a new linear algebra manipulation on electrical impedance spectroscopy measurements to provide real-time information regarding the nature of the tissue surrounding the needle in minimal invasive procedures. Using a Comsol Multiphysics three-dimensional model, a phantom based on ex vivo animal tissue and in vivo animal data, we demonstrate how tissue inhomogeneity can be characterized without any previous knowledge of the electrical properties of the different tissues, except that they should not be linearly dependent on a certain frequency range. This method may have applications in needle biopsies, radiation seeds, or minimally invasive surgery and can reduce the number of computer tomography or magnetic resonance imaging images. We conclude by demonstrating how this mathematical approach can be useful in other applications.

  2. Imaging and finite element analysis: a methodology for non-invasive characterization of aortic tissue. (United States)

    Flamini, Vittoria; Creane, Arthur P; Kerskens, Christian M; Lally, Caitríona


    Characterization of the mechanical properties of arterial tissues usually involves an invasive procedure requiring tissue removal. In this work we propose a non-invasive method to perform a biomechanical analysis of cardiovascular aortic tissue. This method is based on combining medical imaging and finite element analysis (FEA). Magnetic resonance imaging (MRI) was chosen since it presents relatively low risks for human health. A finite element model was created from the MRI images and loaded with systolic physiological pressures. By means of an optimization routine, the structural material properties were changed until average strains matched those measured by MRI. The method outlined in this work produced an estimate of the in situ properties of cardiovascular tissue based on non-invasive image datasets and finite element analysis.

  3. Culture of porcine luteal cells as a substrate for in vitro maturation of porcine cumulus oocyte complexes. Establishment and characterization

    Directory of Open Access Journals (Sweden)

    Teplitz MA


    Full Text Available The aim of this study was to establish and characterize the porcine luteal cells (PLC culture for the subsequent coculture with porcine COC. The final purpose is to promote the oocyte maturation. The PLC was established using corpora lutea obtained from slaughterhouse ovaries. Corpora lutea were dissected and luteal tissue submitted to a mechanical and enzymatic digestion with collagenase IV. The cell suspension was filtered and centrifuged and the cells obtained were diluted in 15 mL of DMEM-F12 supplemented media. Diluted cells were seeded in 3 culture flasks T25, staying in a controlled environment and changing the medium every 2 days. For the analysis and characterization, the cells were assessed by the Nile red staining to detect intracellular lipids, immunocytochemistry (ICC for 3β-hydroxy steroid dehidrogenase (3β-HSD and ELISA for P4 determination. We observed the presence of lipid intracellular droplets. Also, we observed an increase of P4 concentration at 48, 96 y 144 h of primary culture and almost all the cells were positive to the ICC evaluation for 3β-HSD, showing the steroidogenic capacity of the culture cells.

  4. Phenolic content in differentiated tissue cultures of untransformed and Agrobacterium-transformed roots of anise (Pimpinella anisum L.). (United States)

    Andarwulan, N; Shetty, K


    To investigate the role of differentiation of anise tissue cultures on total phenolic and anethole contents, benzylaminopurine- and thidiazuron-induced shoot cultures were generated from roots of the A-8 clonal line and its Agrobacterium rhizogenes-induced genetically transformed derivative JB-10. Embryogenic cultures were induced following 2,4-D treatment. Root cultures were multiplied on hormone-free medium. The effect of proline on differentiation and phenolic synthesis was also investigated. GC/MS studies indicate that anethole was not produced in root or other differentiated cultures. The predominant phenolic metabolite, however, was an anethole precursor, epoxypseudoisoeugenol-2-methylbutyrate (EPB). Total phenolics and EPB contents were highest in root cultures, which also correlated with higher proline content. Embryo and shoot cultures had reduced phenolic level and EPB and proline contents. Antioxidant activity in all differentiating cultures was high on day 60 compared to that on day 30, and there was no significant difference between differentiating tissues. This indicated that antioxidant protection might be linked not only to phenolics but to other nonphenolic metabolites as well.

  5. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda;


    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

  6. Combining scanning haptic microscopy and fibre optic Raman spectroscopy for tissue characterization. (United States)

    Candefjord, S; Murayama, Y; Nyberg, M; Hallberg, J; Ramser, K; Ljungberg, B; Bergh, A; Lindahl, O A


    The tactile resonance method (TRM) and Raman spectroscopy (RS) are promising for tissue characterization in vivo. Our goal is to combine these techniques into one instrument, to use TRM for swift scanning, and RS for increasing the diagnostic power. The aim of this study was to determine the classification accuracy, using support vector machines, for measurements on porcine tissue and also produce preliminary data on human prostate tissue. This was done by developing a new experimental set-up combining micro-scale TRM-scanning haptic microscopy (SHM)-for assessing stiffness on a micro-scale, with fibre optic RS measurements for assessing biochemical content. We compared the accuracy using SHM alone versus SHM combined with RS, for different degrees of tissue homogeneity. The cross-validation classification accuracy for healthy porcine tissue types using SHM alone was 65-81%, and when RS was added it increased to 81-87%. The accuracy for healthy and cancerous human tissue was 67-70% when only SHM was used, and increased to 72-77% for the combined measurements. This shows that the potential for swift and accurate classification of healthy and cancerous prostate tissue is high. This is promising for developing a tool for probing the surgical margins during prostate cancer surgery.

  7. Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture. (United States)

    Jones, Andrew Maxwell Phineas; Saxena, Praveen Kumar


    Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL), thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP), a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.

  8. Mechanical characterization of a customized decellularized scaffold for vascular tissue engineering. (United States)

    Sheridan, W S; Duffy, G P; Murphy, B P


    Several challenges persist when attempting to utilize decellularized tissue as a scaffold for vascular tissue engineering. Namely: poor cell infiltration/migration, excessive culture times associated with repopulating the scaffolds, and the achievement of a quiescent medial layer. In an attempt to create an optimum vascular scaffold, we customized the properties of decellularized porcine carotid arteries by: (i) creating cavities within the medial layer to allow direct injection of cells, and (ii) controlling the amount of collagen digestion to increase the porosity. Histological examination of our customized scaffold revealed a highly porous tissue structure containing consistent medial cavities running longitudinally through the porous scaffold wall. Mechanical testing of the customized scaffold showed that our minimal localized disruption to the ECM does not have a detrimental effect on the bulk mechanical response of the tissue. The results demonstrate that an increased stiffness and reduced distensibility occurs after decellularization when compared to the native tissue, however post scaffold customization we can revert the scaffold tensile properties back to that of the native tissue. This most noteworthy result occurs in the elastin dominant phase of the tensile response of the scaffold, indicating that no disruption has occurred to the elastin network by our decellularization and customization techniques. Additionally, the bulk seeding potential of the customized scaffold was demonstrated by direct injection of human smooth muscle cells through the medial cavities. The optimum cell dispersion was observed in the highest porosity scaffold, with large cell numbers retained within the medial layer after 24 h static culture. In summary, this study presents a novel customized decellularized vascular scaffold that has the capability of bulk seeding the media, and in tandem to this method, the porosity of the scaffold has been increased without compromising the

  9. Establishment and Molecular Characterization of Mesenchymal Stem Cell Lines Derived From Human Visceral & Subcutaneous Adipose Tissues

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    Jyoti Prakash Sutar


    Full Text Available Mesenchymal stem cells (MSCs, are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, myocytes and adipocytes. We utilized adipose tissue as our primary source, since it is a rich source of MSCs as well as it can be harvested using a minimally invasive surgical procedure. Both visceral and subcutaneous adipose tissue (VSAT, SCAT respectively samples were cultured using growth medium without using any substratum for their attachment. We observed growth of mesenchymal like cells within 15 days of culturing. In spite of the absence of any substratum, the cells adhered to the bottom of the petri dish, and spread out within 2 hours. Presently VSAT cells have reached at passage 10 whereas; SCAT cells have reached at passage 14. Morphologically MSCs obtained from visceral adipose tissue were larger in shape than subcutaneous adipose tissue. We checked these cells for presence or absence of specific stem cell molecular markers. We found that VSAT and SCAT cells confirmed their MSC phenotype by expression of specific MSC markers CD 105 and CD13 and absence of CD34 and CD 45 markers which are specific for haematopoietic stem cells. These cells also expressed SOX2 gene confirming their ability of self-renewal as well as expressed OCT4, LIF and NANOG for their properties for pluripotency & plasticity. Overall, it was shown that adipose tissue is a good source of mesenchymal stem cells. It was also shown that MSCs, isolated from adipose tissue are multipotent stem cells that can differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes and liver cells which may open a new era for cell based regenerative therapies for bone, cardiac and liver disorders.

  10. Persistence of the tissue culture origin vaccine for infectious laryngotracheitis virus in commercial chicken flocks in Brazil. (United States)

    Parra, Silvana H Santander; Nuñez, Luis F; Astolfi-Ferreira, Claudete S; Ferreira, Antonio J Piantino


    Infectious laryngotracheitis (ILT) is a respiratory disease of great importance that causes serious economic losses in the poultry industry. Its control is based on biosecurity procedures and vaccination programs that use live attenuated vaccines such as tissue culture origin (TCO), chicken embryo origin (CEO), and vectored vaccines. However, problems have been reported, such as the reversion of virulence, virus latency, and field virus outbreaks. Several molecular techniques have been developed to differentiate between the field and vaccine strains. This study was conducted to determine the presence of infectious laryngotracheitis virus (ILTV) in Brazil from 2012 to 2014. PCR-RFLP (restriction fragment length polymorphism) was used to detect and differentiate ILTV strains; DNA sequencing and predictive RFLP analysis were also used for this purpose. Molecular analysis detected the presence of ILTV in 15 samples that were characterized as strains of TCO vaccine origin. This study showed that the ILTV TCO vaccine strain has been circulating in commercial chicken flocks in Brazil since its introduction during the 2002 outbreak.

  11. A comparative study of three tissue-cultured Dendrobium species and their wild correspondences by headspace gas chromatography–mass spectrometry combined with chemometric methods


    Nai-Dong Chen; Tao You; Jun Li; Li-Tao Bai; Jing-Wen Hao; Xiao-Yuan Xu


    Plant tissue culture technique is widely used in the conservation and utilization of rare and endangered medicinal plants and it is crucial for tissue culture stocks to obtain the ability to produce similar bioactive components as their wild correspondences. In this paper, a headspace gas chromatography–mass spectrometry method combined with chemometric methods was applied to analyze and evaluate the volatile compounds in tissue-cultured and wild Dendrobium huoshanense Cheng and Tang, Dendrob...

  12. Tissue culture of Baiyu chewing cane%白玉蔗组织培养研究

    Institute of Scientific and Technical Information of China (English)

    余坤兴; 杨柳; 刘俊仙; 李松; 方锋学; 杨丽涛; 李杨瑞


    [Objective]Using the Baiyu chewing cane collected from Guigang City, Guangxi as testing material, the main factors impacting Baiyu chewing cane in vitro culture was explored in order to establish the rapid regeneration propagation system. [Method]After using the tissues from Baiyu chewing cane's slightly-tailing leaves as explant callus for bud differentiation culture of callus, adventitious bud proliferation, and plantlet browning prevention and rooting induction were conducted using different culture medium and hormone combinations. [ Result ]Baiyu chewing cane leaf slicing for callus induction was a convenient and effective method. After the whorl sections were connected with the culture medium (MS+2,4-D 3 mg/L, agar 5 g/L, sugar 30 g/L) for 30 d, explants rapidly expanded, the edges became granular, light yellow or yellow, and the embryogenic callus or paste callus displayed uniformed quality. The optimum culture medium for buds differentiation induction in Baiyu chewing cane was MS+6-BA 1.0 mg/L+ NAA0.2 mg/L. For the proliferation of adventitious buds, the optimum effect resulted from the MS+6-BA 3.0 mg/L+KT 0.5 mg/L+NAA 0.6 mg/L+ sugar 30.0 g/L treatment. To prevent browning effects, the PVP and activated carbon tissue culture seedlings had different effects; the 1% activated carbon could effectively inhibit the proliferation of adventitious shoots browning in Baiyu chewing cane culture medium. In the rooting culturing process, the optimal culturing medium was MS+ NAA 0.6 mg/L+ sugar 50.0 g/L + activated carbon 0.1%. [Conclusion]By the orthogonal design test, the regeneration system of Baiyu chewing cane breeding was established in order to speed up the breeding rate of Baiyu chewing cane, improve propagation coefficient, and provide an effective way for capital-saving.%[目的]以广西贵港市白玉蔗为材料,探讨影响白玉蔗离体培养的主要因素,建立其快速再生繁殖体系.[方法]以白玉蔗尾梢心叶为外植体,诱导愈伤组织

  13. A Bayesian approach for characterization of soft tissue viscoelasticity in acoustic radiation force imaging. (United States)

    Zhao, Xiaodong; Pelegri, Assimina A


    Biomechanical imaging techniques based on acoustic radiation force (ARF) have been developed to characterize the viscoelasticity of soft tissue by measuring the motion excited by ARF non-invasively. The unknown stress distribution in the region of excitation limits an accurate inverse characterization of soft tissue viscoelasticity, and single degree-of-freedom simplified models have been applied to solve the inverse problem approximately. In this study, the ARF-induced creep imaging is employed to estimate the time constant of a Voigt viscoelastic tissue model, and an inverse finite element (FE) characterization procedure based on a Bayesian formulation is presented. The Bayesian approach aims to estimate a reasonable quantification of the probability distributions of soft tissue mechanical properties in the presence of measurement noise and model parameter uncertainty. Gaussian process metamodeling is applied to provide a fast statistical approximation based on a small number of computationally expensive FE model runs. Numerical simulation results demonstrate that the Bayesian approach provides an efficient and practical estimation of the probability distributions of time constant in the ARF-induced creep imaging. In a comparison study with the single degree of freedom models, the Bayesian approach with FE models improves the estimation results even in the presence of large uncertainty levels of the model parameters.

  14. Characterization of human arterial tissue affected by atherosclerosis using multimodal nonlinear optical microscopy (United States)

    Baria, Enrico; Cicchi, Riccardo; Rotellini, Matteo; Nesi, Gabriella; Massi, Daniela; Pavone, Francesco S.


    Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. The rupture of an atherosclerotic plaque, resulting in a thrombus, is one of the leading causes of death in the Western World. Preventive assessment of plaque vulnerability is therefore extremely important and can be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires immune-histochemical examination and a morpho-functional approach. Instead, a label-free and non-invasive alternative is provided by nonlinear microscopy. In this study, we combined SHG and FLIM microscopy in order to characterize collagen organization and lipids in human carotid ex vivo tissues affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were processed through image pattern analysis methods (FFT, GLCM). The resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterization of carotid samples and discrimination of different tissue regions. The presented method can be applied for automated classification of atherosclerotic lesions and plaque vulnerability. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in clinical setting.

  15. Effect of echo artifacts on characterization of pulsatile tissues in neonatal cranial ultrasonic movies (United States)

    Fukuzawa, Masayuki; Takahashi, Kazuki; Tabata, Yuki; Kitsunezuka, Yoshiki


    Effect of echo artifacts on characterization of pulsatile tissues has been examined in neonatal cranial ultrasonic movies by characterizing pulsatile intensities with different regions of interest (ROIs). The pulsatile tissue, which is a key point in pediatric diagnosis of brain tissue, was detected from a heartbeat-frequency component in Fourier transform of a time-variation of 64 samples of echo intensity at each pixel in a movie fragment. The averages of pulsatile intensity and power were evaluated in two ROIs: common fan-shape and individual cranial-shape. The area of pulsatile region was also evaluated as the number of pixels where the pulsatile intensity exceeds a proper threshold. The extracranial pulsatile region was found mainly in the sections where mirror image was dominant echo artifact. There was significant difference of pulsatile area between two ROIs especially in the specific sections where mirror image was included, suggesting the suitability of cranial-shape ROI for statistical study on pulsatile tissues in brain. The normalized average of pulsatile power in the cranial-shape ROI exhibited most similar tendency to the normalized pulsatile area which was treated as a conventional measure in spite of its requirement of thresholding. It suggests the potential of pulsatile power as an alternative measure for pulsatile area in further statistical study of pulsatile tissues because it was neither affected by echo artifacts nor threshold.

  16. Detection and characterization of glaucoma-like canine retinal tissues using Raman spectroscopy (United States)

    Wang, Qi; Grozdanic, Sinisa D.; Harper, Matthew M.; Hamouche, Karl; Hamouche, Nicholas; Kecova, Helga; Lazic, Tatjana; Hernandez-Merino, Elena; Yu, Chenxu


    Early detection of pathological changes and progression in glaucoma and other neuroretinal diseases remains a great challenge and is critical to reduce permanent structural and functional retina and optic nerve damage. Raman spectroscopy is a sensitive technique that provides rapid biochemical characterization of tissues in a nondestructive and noninvasive fashion. In this study, spectroscopic analysis was conducted on the retinal tissues of seven beagles with acute elevation of intraocular pressure (AEIOP), six beagles with compressive optic neuropathy (CON), and five healthy beagles. Spectroscopic markers were identified associated with the different neuropathic conditions. Furthermore, the Raman spectra were subjected to multivariate discriminate analysis to classify independent tissue samples into diseased/healthy categories. The multivariate discriminant model yielded an average optimal classification accuracy of 72.6% for AEIOP and 63.4% for CON with 20 principal components being used that accounted for 87% of the total variance in the data set. A strong correlation (R2>0.92) was observed between pattern electroretinography characteristics of AEIOP dogs and Raman separation distance that measures the separation of spectra of diseased tissues from normal tissues; however, the underlining mechanism of this correlation remains to be understood. Since AEIOP mimics the pathological symptoms of acute/early-stage glaucoma, it was demonstrated that Raman spectroscopic screening has the potential to become a powerful tool for the detection and characterization of early-stage disease.

  17. Ultrasonic backscattering in tissue: characterization through Nakagami-generalized inverse Gaussian distribution. (United States)

    Agrawal, Rajeev; Karmeshu


    Ultrasonic tissue characterization through composite probability distributions such as Nakagami-lognormal, Nakagami-gamma, Nakagami-inverse Gaussian has been found to be useful. Such a probabilistic description also depicts heavy tails which arise from multiple scattering in tissue besides local and global variations in scattering cross-sections. A new composite probability distribution, viz. Nakagami-generalized inverse Gaussian distribution (NGIGD) with four parameters is proposed which under different limiting conditions results in approximating the known distributions. A salient aspect of the new distribution is that the probability density function (pdf) of NGIGD variate is available in closed form and is analytically tractable.

  18. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Directory of Open Access Journals (Sweden)

    Purificación Gómez-Abellán

    Full Text Available AIMS: to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V and subcutaneous (S adipose tissue (AT in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX on positive and negative clock genes expression. SUBJECTS AND METHODS: VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2 (n = 6. In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX and AT explants treated with DEX (2 hours were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR. RESULTS: CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements in the SAT (situation not present in VAT. A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues. CONCLUSIONS: 24 h patterns in CLOCK and BMAL1 (positive clock elements and PER2 (negative element mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  19. Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture. (United States)

    Sakakura, Eriko; Eiraku, Mototsugu; Takata, Nozomu


    The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM+KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax+/Chx10+ retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro.

  20. DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs

    Directory of Open Access Journals (Sweden)

    Beheshteh Abouhamzeh


    Full Text Available Objective: Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT. We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b and histone deacetyltransferses (HDAC1, HDAC2, HDAC3 in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4 and acetylation of H3K9 (H3K9ac in BADSCs cultures and different passages in vitro. Materials and Methods: In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR, and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3, 5 (P5 and 7 (P7. Results: The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Conclusion: Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages.

  1. Effect of microcavitary alginate hydrogel with different pore sizes on chondrocyte culture for cartilage tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Lei; Yao, Yongchang [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Wang, Dong-an, E-mail: [National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China); Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Chen, Xiaofeng, E-mail: [School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641 (China); National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006 (China)


    In our previous work, a novel microcavitary hydrogel was proven to be effective for proliferation of chondrocytes and maintenance of chondrocytic phenotype. In present work, we further investigated whether the size of microcavity would affect the growth and the function of chondrocytes. By changing the stirring rate, gelatin microspheres in different sizes including small size (80–120 μm), middle size (150–200 μm) and large size (250–300 μm) were prepared. And then porcine chondrocytes were encapsulated into alginate hydrogel with various sizes of gelatin microspheres. Cell Counting Kit-8 (CCK-8), Live/dead staining and real-time PCR were used to analyze the effect of the pore size on cell proliferation and expression of specific chondrocytic genes. According to all the data, cells cultivated in microcavitary hydrogel, especially in small size, had preferable abilities of proliferation and higher expression of cartilaginous markers including type II collagen, aggrecan and cartilage oligomeric matrix protein (COMP). Furthermore, it was shown by western blot assay that the culture of chondrocytes in microcavitary hydrogel could improve the proliferation of cells potentially by inducing the Erk1/2-MAPK pathway. Taken together, this study demonstrated that chondrocytes favored microcavitary alginate hydrogel with pore size within the range of 80–120 μm for better growth and ECM synthesis, in which Erk1/2 pathway was involved. This culture system would be promising for cartilage tissue engineering. - Highlights: • A novel model with microcavitary structure was set up to study the interaction between cells and materials. • Microcavitary alginate hydrogel could enhance the proliferation of chondrocytes and promote the expression of cartilaginous genes as compared with plain alginate hydrogel. • Cells in microcavitary alginate hydrogel with pore size within the range of 80–120 μm were capable of better growth and ECM synthesis.

  2. Preclinical In vivo Imaging for Fat Tissue Identification, Quantification, and Functional Characterization. (United States)

    Marzola, Pasquina; Boschi, Federico; Moneta, Francesco; Sbarbati, Andrea; Zancanaro, Carlo


    Localization, differentiation, and quantitative assessment of fat tissues have always collected the interest of researchers. Nowadays, these topics are even more relevant as obesity (the excess of fat tissue) is considered a real pathology requiring in some cases pharmacological and surgical approaches. Several weight loss medications, acting either on the metabolism or on the central nervous system, are currently under preclinical or clinical investigation. Animal models of obesity have been developed and are widely used in pharmaceutical research. The assessment of candidate drugs in animal models requires non-invasive methods for longitudinal assessment of efficacy, the main outcome being the amount of body fat. Fat tissues can be either quantified in the entire animal or localized and measured in selected organs/regions of the body. Fat tissues are characterized by peculiar contrast in several imaging modalities as for example Magnetic Resonance Imaging (MRI) that can distinguish between fat and water protons thank to their different magnetic resonance properties. Since fat tissues have higher carbon/hydrogen content than other soft tissues and bones, they can be easily assessed by Computed Tomography (CT) as well. Interestingly, MRI also discriminates between white and brown adipose tissue (BAT); the latter has long been regarded as a potential target for anti-obesity drugs because of its ability to enhance energy consumption through increased thermogenesis. Positron Emission Tomography (PET) performed with (18)F-FDG as glucose analog radiotracer reflects well the metabolic rate in body tissues and consequently is the technique of choice for studies of BAT metabolism. This review will focus on the main, non-invasive imaging techniques (MRI, CT, and PET) that are fundamental for the assessment, quantification and functional characterization of fat deposits in small laboratory animals. The contribution of optical techniques, which are currently regarded with

  3. Preclinical In vivo Imaging for Fat Tissue Identification, Quantification, and Functional Characterization (United States)

    Marzola, Pasquina; Boschi, Federico; Moneta, Francesco; Sbarbati, Andrea; Zancanaro, Carlo


    Localization, differentiation, and quantitative assessment of fat tissues have always collected the interest of researchers. Nowadays, these topics are even more relevant as obesity (the excess of fat tissue) is considered a real pathology requiring in some cases pharmacological and surgical approaches. Several weight loss medications, acting either on the metabolism or on the central nervous system, are currently under preclinical or clinical investigation. Animal models of obesity have been developed and are widely used in pharmaceutical research. The assessment of candidate drugs in animal models requires non-invasive methods for longitudinal assessment of efficacy, the main outcome being the amount of body fat. Fat tissues can be either quantified in the entire animal or localized and measured in selected organs/regions of the body. Fat tissues are characterized by peculiar contrast in several imaging modalities as for example Magnetic Resonance Imaging (MRI) that can distinguish between fat and water protons thank to their different magnetic resonance properties. Since fat tissues have higher carbon/hydrogen content than other soft tissues and bones, they can be easily assessed by Computed Tomography (CT) as well. Interestingly, MRI also discriminates between white and brown adipose tissue (BAT); the latter has long been regarded as a potential target for anti-obesity drugs because of its ability to enhance energy consumption through increased thermogenesis. Positron Emission Tomography (PET) performed with 18F-FDG as glucose analog radiotracer reflects well the metabolic rate in body tissues and consequently is the technique of choice for studies of BAT metabolism. This review will focus on the main, non-invasive imaging techniques (MRI, CT, and PET) that are fundamental for the assessment, quantification and functional characterization of fat deposits in small laboratory animals. The contribution of optical techniques, which are currently regarded with

  4. Ultrasonic characterization of three animal mammary tumors from three-dimensional acoustic tissue models (United States)

    Mamou, Jonathan M.

    This dissertation investigated how three-dimensional (3D) tissue models can be used to improve ultrasonic tissue characterization (UTC) techniques. Anatomic sites in tissue responsible for ultrasonic scattering are unknown, which limits the potential applications of ultrasound for tumor diagnosis. Accurate 3D models of tumor tissues may help identify the scattering sites. Three mammary tumors were investigated: a rat fibroadenoma, a mouse carcinoma, and a mouse sarcoma. A 3D acoustic tissue model, termed 3D impedance map (3DZM), was carefully constructed from consecutive histologic sections for each tumor. Spectral estimates (scatterer size and acoustic concentration) were obtained from the 3DZMs and compared to the same estimates obtained with ultrasound. Scatterer size estimates for three tumors were found to be similar (within 10%). The 3DZMs were also used to extract tissue-specific scattering models. The scattering models were found to allow clear distinction between the three tumors. This distinction demonstrated that UTC techniques may be helpful for noninvasive clinical tumor diagnosis.

  5. Quantitative breast tissue characterization using grating-based x-ray phase-contrast imaging (United States)

    Willner, M.; Herzen, J.; Grandl, S.; Auweter, S.; Mayr, D.; Hipp, A.; Chabior, M.; Sarapata, A.; Achterhold, K.; Zanette, I.; Weitkamp, T.; Sztrókay, A.; Hellerhoff, K.; Reiser, M.; Pfeiffer, F.


    X-ray phase-contrast imaging has received growing interest in recent years due to its high capability in visualizing soft tissue. Breast imaging became the focus of particular attention as it is considered the most promising candidate for a first clinical application of this contrast modality. In this study, we investigate quantitative breast tissue characterization using grating-based phase-contrast computed tomography (CT) at conventional polychromatic x-ray sources. Different breast specimens have been scanned at a laboratory phase-contrast imaging setup and were correlated to histopathology. Ascertained tumor types include phylloides tumor, fibroadenoma and infiltrating lobular carcinoma. Identified tissue types comprising adipose, fibroglandular and tumor tissue have been analyzed in terms of phase-contrast Hounsfield units and are compared to high-quality, high-resolution data obtained with monochromatic synchrotron radiation, as well as calculated values based on tabulated tissue properties. The results give a good impression of the method’s prospects and limitations for potential tumor detection and the associated demands on such a phase-contrast breast CT system. Furthermore, the evaluated quantitative tissue values serve as a reference for simulations and the design of dedicated phantoms for phase-contrast mammography.

  6. Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

    NARCIS (Netherlands)

    van Midwoud, Paul M.; Janse, Arnout; Merema, M.T.; Groothuis, Geny M. M.; Verpoorte, Elisabeth


    Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision cut

  7. Cytoenzymology and 3H-thymidine uptake of retro-ocular connective tissue cultures in experimental endocrino-exophthalmos. (United States)

    Vaida, E; Petrescu, R; Ghinea, E; Stefaneanu, L


    The in vitro retro-ocular connective tissue cultures from guinea pigs with endocrine exophthalmos were studied before and after retro-ocular treatment with cortisol and hyaluronidase. Both cortisol and hyaluronidase inhibited the cell proliferation, the cytoenzymic activities of oxydoreductases, the 3H-thymidine uptake, the number of mitoses and the protein content of cultivated cells.

  8. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F


    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  9. The tissue culture of Rieger begonia%丽格海棠的组织培养

    Institute of Scientific and Technical Information of China (English)

    金雯; 彭佳佳; 秦扬; 刘春; 张黎


    In this experiment,Young Leaves and leaves with petioles of Begonia rieger 'Barkos' and 'Sang Deni' as explants for microprapagation. callus inducing,bud differentiation and proliferation of adventitious bud, rootage inducing were studied under culture media with different concentration of hormones and culture conditions. The result showed: MS+6-BA 0.05mg/L+NAA0.2mg/L is suitable for inducing callus, induction rate of two varieties are 100%and 91.7%.MS+6-BA 0.1mg/L+NAA 0.05mg/L is suitable for bud proliferation, the rate of bud regeneration is 81%.The optimum rooting medium for: 1/2 MS +IBA0.2 mg/L, the rooting rate achieves 100%.And suitable conditions for stage of proliferation of adventitious bud are found out.The study will provide important basis for the rapid propagation of Begonia rieger by tissue culture.%本试验以丽格海棠“巴克斯”和“桑德尼”2个品种的幼嫩叶片、带叶柄叶片为外植体进行组织培养,分别研究不同激素质量浓度以及不同培养条件对丽格海棠愈伤组织诱导、不定芽分化、增殖培养、诱导生根的影响.结果表明:MS+6-BA 0.05 mg/L+NAA 0.2 mg/L适合于愈伤组织的诱导,2个品种的诱导率分别为100%和91.7%. MS+6-BA 0.1 mg/L +NAA 0.05 mg/L适合于不定芽增殖培养基.分化率为81.2%,1/2 MS + IBA0.2 mg/L适合于诱导生根,生根率达100%.同时,研究出增殖阶段外植体最适生长环境,为丽格海棠的组织培养快速繁殖提供了重要依据.

  10. 核桃外植体的组织培养%Tissue Culture of Walnut Explant

    Institute of Scientific and Technical Information of China (English)

    杨海波; 王娟; 周鹏程; 孟利峰; 高涛


    [Objective] The aim was to study the method of tissue culture and rapid propagation of walnut. [ Method] The explains were col lected from stem with axillary buds, the tube propagation of Jinboxiangl was studied. [ Result] The optimum culture medium for germination of the axillary buds: DKW + BA 1.5 mg/L; for differentiation and subculture:DKW + BA 0.4 mg/L + IBA 0.01 mg/L; for rooting;l/2 DKW + IBA 1.0 mg/L, however, rooting efficiency was only 23.3% , this result was not satisfied. The method of taking tool with two steps was ide al, and taking root efficiency reached to 36.7%. [ Conclusion ] The optimal culture conditions of rapid propagation of Jinboxiang 1 walnut were selected, which laid the foundation for constructing rapid propagation and amplifying walnut planting scale.%[目的]研究核桃组织培养与快速繁殖的方法.[方法]以“金薄香”1号带腋芽的茎段为外植体进行试管繁殖.[结果]腋芽萌生最佳培养基:DKW+BA 1.5 mg/L;分化及继代最佳培养基:DKW+ BA 0.4 mg/L+ IBA 0.01 mg/L;生根最佳培养基:1/2DKW+ IBA 1.0mg/L,不过生根率只有23.3%,效果不太理想;二步生根法效果不错,生根率可达到36.7%.[结论]该研究筛选获得适宜“金薄香”1号核桃快繁的最佳培养条件,为建立核桃快繁体系、扩大核桃苗繁育规模奠定基础.

  11. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Directory of Open Access Journals (Sweden)

    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  12. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants. (United States)

    Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz


    Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

  13. Characterization of water molecular state in in-vivo thick tissues using diffuse optical spectroscopic imaging (United States)

    Chung, So Hyun

    Structural changes in water molecules are related to physiological, anatomical and pathological properties of tissues. Near infrared (NIR) optical absorption methods are sensitive to water; however, detailed characterization of water in thick tissues is difficult to achieve because subtle spectral shifts can be obscured by multiple light scattering. In the NIR, a water absorption peak is observed around 975 nm. The precise NIR peak's shape and position are highly sensitive to water molecular disposition. A bound water index (BWI) was developed that quantifies the spectral shift and shape changes observed in tissue water absorption spectra measured by broadband diffuse optical spectroscopic imaging (DOSI). DOSI quantitatively measures light absorption and scattering spectra in cm-deep tissues and therefore reveals bound water spectral shifts. BWI as a water state index was validated by comparing broadband DOSI to MRI and a conductivity cell using bound water phantoms. Non-invasive BWI measurements of malignant and normal tissues in 18 subjects showed a significantly higher fraction of free water in malignant tissues (pbreast cancer patients. The BWI and ADC correlated (R=0.8, p=<0.01) and both parameters decreased with increasing bulk water content in cancer tissues. Although BWI and ADC are positively correlated in vivo, BWI appears to be more sensitive to free water in the extracellular matrix while ADC reflects increased tumor cellularity. The relationship between ADC, BWI and bulk water concentration suggests that both parameters have potential for assessing tumor histopathological grade. My results confirm the importance of water as a critical tissue component that can potentially provide unique insight into the molecular pathophysiology of cancer.

  14. Qualitative and quantitative analysis of anthraquinone derivatives in rhizomes of tissue culture-raised Rheum emodi Wall. plants. (United States)

    Malik, Sonia; Sharma, Nandini; Sharma, Upendra K; Singh, Narendra P; Bhushan, Shashi; Sharma, Madhu; Sinha, Arun K; Ahuja, Paramvir S


    This paper presents quantification of five anthraquinone derivatives (emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion) in rhizomes of hardened micro-propagated Rheum emodi plants using high-performance liquid chromatography (HPLC). Aseptic shoot cultures were raised using rhizome buds. Shoot multiplication occurred in both agar gelled and liquid Murashige and Skoog (MS) medium supplemented with 10.0 microM 6-benzylaminopurine (BAP) and 5.0 microM indole-3-butyric acid (IBA). Rooted plantlets obtained on plant growth regulator (PGR)-free medium were transferred to soil with 92% survival. HPLC analysis revealed the presence of five anthraquinone derivatives: emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion in rhizomes of tissue culture-raised plants. Only emodin glycoside (1) and chrysophanol glycoside (2) were present in 6-month-old hardened tissue cultured plants. In addition, the other three derivatives (emodin (3), chrysophanol (4) and physcion (5)) were also detected after 9 months.

  15. Effects of space environment on biological characters of tissue cultured rose seedlings

    Institute of Scientific and Technical Information of China (English)

    XUE Huai; LIU Min; LU Jinying; PAN Yi; ZHANG Chunhua


    Tissue cultured rose seedlings were carried into space by SHENZHOU-4 spacecraft and then used as the experimental material to investigate effects of the space environmental conditions on morphology, cytology, physiology and molecular biology of the seedlings. After loaded on the space flight, the plant's height, number of leaves, and fresh weight per seedling were all increased significantly compared to the ground controls. The content of chlorophyll was basically unchanged. In some cells, the ultrastructural changes involved twist, contraction and deformation of cell wall, curvature and loose arrangement of lamellae of some chloroplasts, and a significant increase in number of starch grains per chloroplast. In addition, the number of mitochondria increased, but some mitochondrial outer membrane broke, and some mitochondrial cristae disappeared. The activities of the defense enzymes, such as superoxide dismutase, peroxidase and catalyse, in rose leaves increased and the content of malondialdehyde decreased. In the RAPD analysis with 40 10-mer primers, 36 primers generated 148 DNA bands from both of the space flight treated seedlings and the ground controls, and five primers amplified polymorphic products. The rate of DNA variation was 6.34 %.

  16. Correlation of metabolic changes of infected paulownia tissue culture with PWB-MLO pathogenic mechanism

    Institute of Scientific and Technical Information of China (English)

    田国忠; 黄钦才; 袁巧平; 张锡津


    Paulownia sp.tissue culture plantlets infected with paulownia witches’ broom mycoplasma-likeorganism (PWB-MLO) expressed a typical witches’ broom symptom.The results determined by transmissionelectron microscopy and DAPI staining fluorescence microscopy showed that the severity of the symptom wasdirectly related with MLO concentration in the sieve tubes of the phloem.The results of the analysis ofendogenous hormones and phenolic compounds by HPLC revealed that in all cases,the free IAA content ofthe diseased plant apparently lower than that of the healthy,and the free IAA concentration had strikinglypositive correlation with catechol concentration changes (r=0.90,n= 11).The changes of cytokinin (Z +ZR,iPA),GA3 and ABA were shown less relation with MLO infection.The differences of peroxidase andisoenzyme,IAA oxidase and phenylanine ammonia lyase (PAL) between the extract of infected plants andthat of uninfected as well as different effects of NAA and catechol additions on MS media could further re-flect the nature of the relationship and the interaction among MLOs,catechol and the free IAA concentration.

  17. Plant cell, tissue and organ culture: the most flexible foundations for plant metabolic engineering applications. (United States)

    Ogita, Shinjiro


    Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering in target plants are discussed mainly from the phenotypic view point.

  18. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration. (United States)

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P


    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF.

  19. Tissue culture and regeneration of an antimalarial plant, Artemisia sieberi Besser

    Directory of Open Access Journals (Sweden)

    A. Sharafi


    Full Text Available WHO recommends artemisinin-based combination therapies (ACTs as the most effective choice to treat malaria. For developing transgenic plants with high accumulation of artemisinin (by introducing genes encoding enzymes which regulate the biosynthetic pathway of artemisinin, an efficient protocol for tissue culture and plant regeneration is necessary. In the present study, leaf explants of Artemisia sieberi were cultivated in Murashige & Skoog based medium supplemented by combination of different plant growth regulators including 6-benzyl-aminopurine (BA, α-naphthalene-acetic acid (NAA, indole-3-acetic acid (IAA, picloram (Pic and 2,4-dichlorophenoxyacetic acid (2,4-D. The highest frequency of shoot induction was obtained on MS medium supplemented with 2 mg/L BA plus 0.05 mg/L NAA (95% regeneration and MS medium supplemented with 2 mg/L BA plus 0.5 mg/L IAA (85% regeneration. Rooting was obtained on MS medium supplemented with 0.05 mg/L NAA. The present study has revealed a simple, reliable, rapid and high efficient regeneration system for A. sieberi Besser as a source of artemisinin in short period via adventitious shoot induction procedure.

  20. Effects of Nd:YAG laser radiation in cultured porcine vertebral disc tissue (United States)

    Thal, Dietmar R.; Werkmann, Klaus; Leheta, Fouad; Schober, Ralf; Ulrich, Peter


    Nd:Yag laser radiation is used for the treatment of protrusion of intervertebral discs. It is known that laser radiation leads to coagulation, vaporization and carbonization of the disk. Little is known about the early changes in vertebral discs after laser radiation. Therefore, we exposed cadaveric porcine vertebral discs by Nd:YAG laser radiation immediately after death. The discs were quartered and either formalin fixed after laser radiation or kept in culture for 1, 4 and 7 days and then formalin fixed. Immunohistochemistry was performed with antibodies directed against vimentin and amyloid precursor protein (APP). Results showed a jerky leak of notochordial remnant cells and mucopolysaccharides at the distal end of the application needle during laser radiation, which was interpreted as a bursting extrusion of damaged but not vaporized tissue. Histology and immunohistochemistry revealed an incomplete loss of nucleus pulposus and a large, almost complete necrosis of the notochordial remnant cells. In surviving notochordial remnant cells after laser radiation a slight increase of vimentin and APP could be seen without any other cellular reactions. The annulus fibrosus showed no significant changes except a defect with a small necrosis zone at the site of the application needle. Therefore, it can be concluded that Nd:YAG laser radiation leads to an increased volume reduction by the leak of nucleus pulposus and to a slight cellular reaction of surviving notochordal remnant cells detectable by vimentin and APP increase.

  1. Comparison of regeneration potentials in tissue cultures of primitive and cultivated tomato species (Lycopersicon sp.

    Directory of Open Access Journals (Sweden)

    M. Lech


    Full Text Available Regeneration capacities of two tomato cultivars: Potentat and Rutgers, and of three accessions of wild tomato species: Lycopersicon peruvianum PI 128650, L. peruvianum var. dentatum PI 128655 and L. glandulosum were studied using an universal medium suitable for regeneration of those plants from leaf pieces in tissue culture. Fragments of leaf blades were taken from plants raised in greenhouse conditions and placed on a modified MS medium containing 0.3 mg/l IAA and 3.0 mg/l BAP solidified with 1% agar. The explants were transferred every 4-5 weeks on fresh medium of the same composition. It was shown that all the three primitive tomato species revealed much higher multiplication coefficients than the two cultivars. Appropriate values were: 11 - for L. glandulosum, 8 - for L. peruvianum, 7 - for L. peruvianum var. dentatum, 4 - for L. esculentum cv. Potentat and 2 - cv. Rutgers. Completely regenerated plants were obtained from all the tested species, but organogenesis occurred almost two weeks earlier in wild tomatoes than in the culitivated varieties of L. esculentum.

  2. Mesenchymal Stem/Stromal Cells from Discarded Neonatal Sternal Tissue: In Vitro Characterization and Angiogenic Properties

    Directory of Open Access Journals (Sweden)

    Shuyun Wang


    Full Text Available Autologous and nonautologous bone marrow mesenchymal stem/stromal cells (MSCs are being evaluated as proangiogenic agents for ischemic and vascular disease in adults but not in children. A significant number of newborns and infants with critical congenital heart disease who undergo cardiac surgery already have or are at risk of developing conditions related to inadequate tissue perfusion. During neonatal cardiac surgery, a small amount of sternal tissue is usually discarded. Here we demonstrate that MSCs can be isolated from human neonatal sternal tissue using a nonenzymatic explant culture method. Neonatal sternal bone MSCs (sbMSCs were clonogenic, had a surface marker expression profile that was characteristic of bone marrow MSCs, were multipotent, and expressed pluripotency-related genes at low levels. Neonatal sbMSCs also demonstrated in vitro proangiogenic properties. Sternal bone MSCs cooperated with human umbilical vein endothelial cells (HUVECs to form 3D networks and tubes in vitro. Conditioned media from sbMSCs cultured in hypoxia also promoted HUVEC survival and migration. Given the neonatal source, ease of isolation, and proangiogenic properties, sbMSCs may have relevance to therapeutic applications.

  3. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. (United States)

    Knight, Eleanor; Przyborski, Stefan


    Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.

  4. ROC analysis of ultrasound tissue characterization classifiers for breast cancer diagnosis. (United States)

    Gefen, Smadar; Tretiak, Oleh J; Piccoli, Catherine W; Donohue, Kevin D; Petropulu, Athina P; Shankar, P Mohana; Dumane, Vishruta A; Huang, Lexun; Kutay, M Alper; Genis, Vladimir; Forsberg, Flemming; Reid, John M; Goldberg, Barry B


    Breast cancer diagnosis through ultrasound tissue characterization was studied using receiver operating characteristic (ROC) analysis of combinations of acoustic features, patient age, and radiological findings. A feature fusion method was devised that operates even if only partial diagnostic data are available. The ROC methodology uses ordinal dominance theory and bootstrap resampling to evaluate A(z) and confidence intervals in simple as well as paired data analyses. The combined diagnostic feature had an A(z) of 0.96 with a confidence interval of at a significance level of 0.05. The combined features show statistically significant improvement over prebiopsy radiological findings. These results indicate that ultrasound tissue characterization, in combination with patient record and clinical findings, may greatly reduce the need to perform biopsies of benign breast lesions.

  5. Multi-scale mechanical characterization of scaffolds for heart valve tissue engineering. (United States)

    Argento, G; Simonet, M; Oomens, C W J; Baaijens, F P T


    Electrospinning is a promising technology to produce scaffolds for cardiovascular tissue engineering. Each electrospun scaffold is characterized by a complex micro-scale structure that is responsible for its macroscopic mechanical behavior. In this study, we focus on the development and the validation of a computational micro-scale model that takes into account the structural features of the electrospun material, and is suitable for studying the multi-scale scaffold mechanics. We show that the computational tool developed is able to describe and predict the mechanical behavior of electrospun scaffolds characterized by different microstructures. Moreover, we explore the global mechanical properties of valve-shaped scaffolds with different microstructural features, and compare the deformation of these scaffolds when submitted to diastolic pressures with a tissue engineered and a native valve. It is shown that a pronounced degree of anisotropy is necessary to reproduce the deformation patterns observed in the native heart valve.

  6. X-ray scattering for the characterization of lyophilized breast tissue samples (United States)

    Elshemey, Wael M.; Mohamed, Fayrouz S.; Khater, Ibrahim M.


    This work investigates the possibility of characterizing breast cancer by measuring the X-ray scattering profiles of lyophilized excised breast tissue samples. Since X-ray scattering from water-rich tissue is dominated by scattering from water, the removal of water by lyophilization would enhance the characterization process. In the present study, X-ray scattering profiles of 22 normal, 22 malignant and 10 benign breast tissue samples are measured. The cut-offs of scatter diagrams, sensitivity, specificity and diagnostic accuracy of three characterization parameters (full width at half maximum (FWHM) for the peak at 1.1 nm-1, area under curve (AUC), and ratio of 1st to 2nd scattering peak intensities (I1/I2%)) are calculated and compared to the data from non-lyophilized samples. Results show increased sensitivity (up to 100%) of the present data on lyophilized breast tissue samples compared to previously reported data for non-lyophilized samples while the specificity (up to 95.4%), diagnostic accuracy (up to 95.4%) and receiver operating characteristic (ROC) curve values (up to 0.9979) for both sets of data are comparable. The present study shows significant differences between normal samples and each of malignant and benign samples. Only subtle differences exist between malignant and benign lyophilized breast tissue samples where FWHM=0.7±0.1 and 0.8±0.3, AUC=1.3±0.2 and 1.4±0.2 and I1/I2%=44.9±11.0 and 52.4±7.6 for malignant and benign samples respectively.

  7. TU-CD-207-01: Characterization of Breast Tissue Composition Using Spectral Mammography

    Energy Technology Data Exchange (ETDEWEB)

    Ding, H; Cho, H; Kumar, N; Sennung, D; Ng, A Lam; Molloi, S [Department of radiological scicens, University of California, Irvine, CA (United States)


    Purpose: To investigate the feasibility of characterizing the chemical composition of breast tissue, in terms of water and lipid, by using spectral mammography in simulation and postmortem studies. Methods: Analytical simulations were performed to obtain low- and high-energy signals of breast tissue based on previously reported water, lipid, and protein contents. Dual-energy decomposition was used to characterize the simulated breast tissue into water and lipid basis materials and the measured water density was compared to the known value. In experimental studies, postmortem breasts were imaged with a spectral mammography system based on a scanning multi-slit Si strip photon-counting detector. Low- and high-energy images were acquired simultaneously from a single exposure by sorting the recorded photons into the corresponding energy bins. Dual-energy material decomposition of the low- and high-energy images yielded individual pixel measurements of breast tissue composition in terms of water and lipid thicknesses. After imaging, each postmortem breast was chemically decomposed into water, lipid and protein. The water density calculated from chemical analysis was used as the reference gold standard. Correlation of the water density measurements between spectral mammography and chemical analysis was analyzed using linear regression. Results: Both simulation and postmortem studies showed good linear correlation between the decomposed water thickness using spectral mammography and chemical analysis. The slope of the linear fitting function in the simulation and postmortem studies were 1.15 and 1.21, respectively. Conclusion: The results indicate that breast tissue composition, in terms of water and lipid, can be accurately measured using spectral mammography. Quantitative breast tissue composition can potentially be used to stratify patients according to their breast cancer risk.

  8. Characterization of biomechanical properties of agar based tissue mimicking phantoms for ultrasound stiffness imaging techniques. (United States)

    Manickam, Kavitha; Machireddy, Ramasubba Reddy; Seshadri, Suresh


    Pathological changes of the body have been observed to change the mechanical properties of soft tissue types which can be imaged by ultrasound elastography. Though initial clinical results using ultrasound elastography in detection of tumors are promising, quantification of signal to noise ratio, resolution and strain image patterns are the best achieved under a controlled study using phantoms with similar biomechanical properties of normal and abnormal tissues. The purpose of this work is to characterize the biomechanical properties of agar based tissue mimicking phantoms by varying the agar concentration from 1.7 to 6.6% by weight and identify the optimum property to be used in classification of cancerous tissues. We performed quasi-static uniaxial compression test under a strain rate of 0.5mm/min up to 15% strain and measured Young's modulus of phantom samples which are from 50kPa to 450kPa. Phantoms show nonlinear stress-strain characteristics at finite strain which were characterized using hyperelastic parameters by fitting Neo-Hookean, Mooney Rivlin, Ogden and Veronda Westmann models. We also investigated viscoelastic parameters of the samples by conducting oscillatory shear rheometry at various precompression levels (2-5%). Loss modulus values are always less than storage modulus which represents the behavior of soft tissues. The increase in agar concentration increases the shear modulus of the samples as well as decreases the linear viscoelastic region. The results suggest that dynamic shear modul are more promising than linear and nonlinear elastic modul in differentiation of various classes of abnormal tissues.

  9. New methodology for mechanical characterization of human superficial facial tissue anisotropic behaviour in vivo. (United States)

    Then, C; Stassen, B; Depta, K; Silber, G


    Mechanical characterization of human superficial facial tissue has important applications in biomedical science, computer assisted forensics, graphics, and consumer goods development. Specifically, the latter may include facial hair removal devices. Predictive accuracy of numerical models and their ability to elucidate biomechanically relevant questions depends on the acquisition of experimental data and mechanical tissue behavior representation. Anisotropic viscoelastic behavioral characterization of human facial tissue, deformed in vivo with finite strain, however, is sparse. Employing an experimental-numerical approach, a procedure is presented to evaluate multidirectional tensile properties of superficial tissue layers of the face in vivo. Specifically, in addition to stress relaxation, displacement-controlled multi-step ramp-and-hold protocols were performed to separate elastic from inelastic properties. For numerical representation, an anisotropic hyperelastic material model in conjunction with a time domain linear viscoelasticity formulation with Prony series was employed. Model parameters were inversely derived, employing finite element models, using multi-criteria optimization. The methodology provides insight into mechanical superficial facial tissue properties. Experimental data shows pronounced anisotropy, especially with large strain. The stress relaxation rate does not depend on the loading direction, but is strain-dependent. Preconditioning eliminates equilibrium hysteresis effects and leads to stress-strain repeatability. In the preconditioned state tissue stiffness and hysteresis insensitivity to strain rate in the applied range is evident. The employed material model fits the nonlinear anisotropic elastic results and the viscoelasticity model reasonably reproduces time-dependent results. Inversely deduced maximum anisotropic long-term shear modulus of linear elasticity is G∞,max(aniso)=2.43kPa and instantaneous initial shear modulus at an

  10. A novel gellan-PVA nanofibrous scaffold for skin tissue regeneration: Fabrication and characterization. (United States)

    Vashisth, Priya; Nikhil, Kumar; Roy, Partha; Pruthi, Parul A; Singh, Rajesh P; Pruthi, Vikas


    In this investigation, we have introduced novel electrospun gellan based nanofibers as a hydrophilic scaffolding material for skin tissue regeneration. These nanofibers were fabricated using a blend mixture of gellan with polyvinyl alcohol (PVA). PVA reduced the repulsive force of resulting solution and lead to formation of uniform fibers with improved nanostructure. Field emission scanning electron microscopy (FESEM) confirmed the average diameter of nanofibers down to 50 nm. The infrared spectra (IR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis evaluated the crosslinking, thermal stability and highly crystalline nature of gellan-PVA nanofibers, respectively. Furthermore, the cell culture studies using human dermal fibroblast (3T3L1) cells established that these gellan based nanofibrous scaffold could induce improved cell adhesion and enhanced cell growth than conventionally proposed gellan based hydrogels and dry films. Importantly, the nanofibrous scaffold are biodegradable and could be potentially used as a temporary substrate/or biomedical graft to induce skin tissue regeneration.

  11. Purification and characterization of α-L-fucosidase from human primary hepatocarcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Jie Qian; Ju-Sheng Lin


    AIM: To purify and characterize α-L-fucosidase fromhuman liver cancer tissue and to detect the localizationof α-L-fucosidase in tumor tissue.METHODS: Cation exchange chromatography onCM-52 and ultrafiltration were used to separate α-L-fucosidase (AFU) from crude extract of liver cancertissue. 4-methylumbelliferyl-α-L-fucopyranoside wasused as a fluorescent substrate to quantify the purifiedAFU activity in each step. A polyclonal antibody (pAb)against the purified AFU was obtained by anion exchangechromatography on DEAE-52 after ammonium sulfatefractionation and ultrafiltration. Immuohistochemicalstaining was used to observe the expression of AFU inmalignant and adjacent liver tissues.RESULTS: Human α-L-fucosidase was purified 74-fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of molecularweight of 55 Ku. The specific activity of AFU in pooledfraction by chromatography was 10085 IU/mg. Westernblot analysis indicated that the pAb could recognizeone protein band of molecular weight of 55 Ku. Theexpression of AFU was observed in cytoplasm membraneof liver cancer tissue but not in that of adjacent tissue.CONCLUSION: The purified α-L-fucosidase from primaryhepatocarcinoma (PHC) is different in its properties fromα-L-fucosidase in human other organs. The polyclonalantibody prepared in this experiment can be applied tothe diagnosis of PHC.

  12. Magnetization transfer characterization of hypertensive cardiomyopathy: significance of tissue water content. (United States)

    Scholz, T D; Ceckler, T L; Balaban, R S


    Magnetization transfer measurements offer the potential for specific noninvasive tissue characterization. The goal of the present study was to determine if changes in magnetization transfer would accompany the myocardial remodeling that occurs with hypertrophic cardiomyopathy. Using 40-week spontaneously hypertensive rat (SHR) myocardium, T1, T2, and T1 in the presence of off-resonance irradiation (T1sat) were found to be greater compared to Wistar-Kyoto (WKY) controls. The pseudo-first order rate constant of magnetization transfer (kfor) was less in 40-week SHR compared with WKY while the ratio of equilibrium magnetization in the presence and absence of off-resonance irradiation (MsIM0) was not different. The extent to which observed interspecies differences in tissue water content affected these parameters was investigated by dehydrating normal and hypertrophic myocardium. Significant correlations found between tissue water content and T1, T2, T1sat and kfor, but not MsIM0, suggested changes in tissue water dominated the observed interspecies differences in relaxation parameters. Thus, ventricular remodeling in hypertrophic cardiomyopathy does not alter magnetization transfer though the accompanying change in tissue water content influences water proton relaxation.

  13. Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure. (United States)

    Sandberg, Dustin C; Battista, Lorna J; Arnold, A Elizabeth


    Most studies of endophytic symbionts have focused on terrestrial plants, neglecting the ecologically and economically important plants present in aquatic ecosystems. We evaluated the diversity, composition, host and tissue affiliations, and geographic structure of fungal endophytes associated with common aquatic plants in lentic waters in northern Arizona, USA. Endophytes were isolated in culture from roots and photosynthetic tissues during two growing seasons. A total of 226 isolates representing 60 putative species was recovered from 9,600 plant tissue segments. Although isolation frequency was low, endophytes were phylogenetically diverse and species-rich. Comparisons among the most thoroughly sampled species and reservoirs revealed that isolation frequency and diversity did not differ significantly between collection periods, among species, among reservoirs, or as a function of depth. However, community structure differed significantly among reservoirs and tissue types. Phylogenetic analyses of a focal genus (Penicillium) corroborated estimates of species boundaries and informed community analyses, highlighting clade- and genotype-level affiliations of aquatic endophytes with both sediment- and waterborne fungi, and endophytes of proximate terrestrial plants. Together these analyses provide a first quantitative examination of endophytic associations in roots and foliage of aquatic plants and can be used to optimize survey strategies for efficiently capturing fungal biodiversity at local and regional scales.


    Ju, Weina; Wu, Jiang; Pritz, Michael B.; Khanna, Rajesh


    Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarization-induced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist. PMID:24260711

  15. Fabrication and Characterization of Spongy Denuded Amniotic Membrane Based Scaffold for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Ehsan Taghiabadi


    Full Text Available Objective: As a biological tissue material, amniotic membrane (AM has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D spongy scaffold made of the extracellular matrix (ECM of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. Materials and Methods: In this experimental study, cellular components of human amniotic membrane (HAM were removed with 0.03% (w/v sodium dodecyl sulphate (SDS. Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs, collagen, and deoxyribonucleic acid (DNA. To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M with pepsin (1 mg/ml. In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl carbodiimide hydrochloride (EDC/N-hydroxysuccinimide (NHS cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scafolds were cytocompatible. Results: Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml. Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg.With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the

  16. Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue (United States)

    Luo, Wei; Hu, Qiang; Wang, Dan; Deeb, Kristin K.; Ma, Yingyu; Morrison, Carl D.; Liu, Song; Johnson, Candace S.; Trump, Donald L.


    Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissues obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small

  17. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications. (United States)

    Neelakandan, Anjanasree K; Wang, Kan


    In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.

  18. Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.). (United States)

    Lees, G L


    Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.

  19. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station. (United States)

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid; Grivel, Jean-Charles


    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

  20. Synthesis, characterization and antioxidant activity of a novel electroactive and biodegradable polyurethane for cardiac tissue engineering application

    Energy Technology Data Exchange (ETDEWEB)

    Baheiraei, Nafiseh [Department of Tissue Engineering, School of Advanced Medical Technologies, Tehran University of Medical Sciences, 1417755469 Tehran (Iran, Islamic Republic of); Yeganeh, Hamid, E-mail: [Department of Polyurethane, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of); Ai, Jafar [Department of Tissue Engineering, School of Advanced Medical Technologies, Tehran University of Medical Sciences, 1417755469 Tehran (Iran, Islamic Republic of); Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Gharibi, Reza [Department of Polyurethane, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115, Tehran (Iran, Islamic Republic of); Azami, Mahmoud; Faghihi, Faezeh [Department of Tissue Engineering, School of Advanced Medical Technologies, Tehran University of Medical Sciences, 1417755469 Tehran (Iran, Islamic Republic of)


    There has been a growing trend towards applying conducting polymers for electrically excitable cells to increase electrical signal propagation within the cell-loaded substrates. A novel biodegradable electroactive polyurethane containing aniline pentamer (AP-PU) was synthesized and fully characterized by spectroscopic methods. To tune the physico-chemical properties and biocompatibility, the AP-PU was blended with polycaprolactone (PCL). The presence of electroactive moieties and the electroactivity behavior of the prepared films were confirmed by UV–visible spectroscopy and cyclic voltammetry. A conventional four probe analysis demonstrated the electrical conductivity of the films in the semiconductor range (∼ 10{sup −5} S/cm). MTT assays using L929 mouse fibroblast and human umbilical vein endothelial cells (HUVECs) showed that the prepared blend (PB) displayed more cytocompatibility compared with AP-PU due to the introduction of a biocompatible PCL moiety. The in vitro cell culture also confirmed that PB was as supportive as tissue culture plate. The antioxidant activity of the AP-PU was proved using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay by employing UV–vis spectroscopy. In vitro degradation tests conducted in phosphate-buffered saline, pH 7.4 and pH 5.5, proved that the films were also biodegradable. The results of this study have highlighted the potential application of this bioelectroactive polyurethane as a platform substrate to study the effect of electrical signals on cell activities and to direct desirable cell function for tissue engineering applications. - Highlights: • Straight forward methodology for synthesis of electroactive polyurethane • Biodegradability and non-toxicity through proper selection of starting materials • Supporting cell proliferation and attachment combined with antioxidant property.

  1. Characterization of a novel rat cholangiocarcinoma cell culture model-CGCCA

    Institute of Scientific and Technical Information of China (English)

    Chun-Nan Yeh; Kun-Ju Lin; Tsung-Wen Chen; Ren-Ching Wu; Lee-Cheng Tsao; Ying-Tzu Chen; Wen-Hui Weng; Miin-Fu Chen


    AIM: To characterize a culture model of rat CCA cells, which were derived from a transplantable TTA-induced CCA and designated as Chang Gung CCA (CGCCA). METHODS: The CGCCA cells were cultured at in vitro passage 12 times on a culture dish in DMEM medium. To measure the doubling time, 103 cells were plated in a 96-well plate containing the growth medium. The cells were harvested 4 to 10 d after seeding, and a standard MTT assay was used to measure the growth. The phenotype of CACCA cell and xenograft was determined by immunohistochemical study. We also determine the chromosomal alterations of CGCCA, G-banding and spectral karyotyping studies were performed. The CGCCA cell line was transplanted into the nude mice for examining its tumorigenicity. 2-Deoxy-2-(18F)fluoro-Dglucose (FDG) autoradiography was also performed to evaluate the FDG uptake of the tumor xenograft. RESULTS: The doubling time for the CGCCA cell line was 32 h. After transplantation into nude mice, FDG autoradiography showed that the tumors formed at the cell transplantation site had a latency period of 4-6 wk with high FDG uptake excluding necrosis tissue. Moreover, immunohistochemical staining revealed prominent cytoplasmic expression of c-erb-B2, CK19, c-Met, COX-Ⅱ, EGFR, MUC4, and a negative expression of K-ras. All data confirmed the phenotypic features of the CGCCA cell line coincide with the xenograft mice tumors, indicating cells containing the tumorigenicity of CCA originated from CCA. In addition, karyotypic banding analysis showed that the diploid (2n) cell status combines with ring and giant rod marker chromosomes in these clones; either both types simultaneously appeared or only one type of marker chromosome in a pair appeared in a cell. The major materials contained in the marker chromosome were primarily identified from chromosome 4. CONCLUSION: The current CGCCA cell line may be used as a non-K-ras effect CCA model and to obtain information and reveal novel pathways for CCA. Further

  2. Characterization of cyclic AMP accumulation in cultured human corpus cavernosum smooth muscle cells. (United States)

    Palmer, L S; Valcic, M; Melman, A; Giraldi, A; Wagner, G; Christ, G J


    Intracavernous pharmacotherapy relies heavily on the use of vasoactive agents which act by increasing intracellular cAMP levels in human corpus cavernosum smooth muscle. Yet little is known about the cAMP generating system in this tissue, and how it may affect observed patient variability. Thus, the goal of these studies was to better characterize the biochemistry of cAMP formation in human corpus cavernosum smooth muscle, and thus provide more insight into the mechanisms of corporal smooth muscle relaxation in vivo. We studied both receptor and nonreceptor mediated increases in cAMP formation in short-term cultures of human corpus cavernosum smooth muscle cells. Both isoproterenol (ISO) and prostaglandin E1 (PGE1) produced concentration-dependent increases in cAMP, but histamine, serotonin and vasoactive intestinal polypeptide did not. Forskolin, a relatively specific activator of adenylate cyclase, was also a potent stimulant of cAMP formation in these cells. Moreover, there was a direct correlation between the degree of forskolin-induced cAMP accumulation in cultured corporal smooth muscle cells and the magnitude of the forskolin-induced relaxation response of precontracted isolated corporal smooth muscle strips. Prostaglandin E1 and ISO concentration response curves (CRCs) were then assayed in the absence and presence of subthreshold forskolin (0.1 microM.). In the presence of forskolin, the calculated maximal PGE1-induced cAMP accumulation (Emax) was significantly greater than that elicited by PGE1 alone, ISO alone, or ISO + forskolin (p protocol was used to demonstrate that both 80:20 and 70:30 FMRs (but not 95:5 or 90:10), were associated with significantly greater cAMP Emax values than that observed for PGE1 alone (p < or = 0.01). These data provide direct evidence that the degree of cAMP formation in cultured corporal smooth muscle cells is strongly correlated with the magnitude of relaxation of isolated corporal smooth muscle strips. In addition, since

  3. Spatiotemporal Characterization of Extracellular Matrix Microstructures in Engineered Tissue: A Whole-Field Spectroscopic Imaging Approach. (United States)

    Xu, Zhengbin; Ozcelikkale, Altug; Kim, Young L; Han, Bumsoo


    Quality and functionality of engineered tissues are closely related to the microstructures and integrity of their extracellular matrix (ECM). However, currently available methods for characterizing ECM structures are often labor-intensive, destructive, and limited to a small fraction of the total area. These methods are also inappropriate for assessing temporal variations in ECM structures. In this study, to overcome these limitations and challenges, we propose an elastic light scattering approach to spatiotemporally assess ECM microstructures in a relatively large area in a nondestructive manner. To demonstrate its feasibility, we analyze spectroscopic imaging data obtained from acellular collagen scaffolds and dermal equivalents as model ECM structures. For spatial characterization, acellular scaffolds are examined after a freeze/thaw process mimicking a cryopreservation procedure to quantify freezing-induced structural changes in the collagen matrix. We further analyze spatial and temporal changes in ECM structures during cell-driven compaction in dermal equivalents. The results show that spectral dependence of light elastically backscattered from engineered tissue is sensitively associated with alterations in ECM microstructures. In particular, a spectral decay rate over the wavelength can serve as an indicator for the pore size changes in ECM structures, which are at nanometer scale. A decrease in the spectral decay rate suggests enlarged pore sizes of ECM structures. The combination of this approach with a whole-field imaging platform further allows visualization of spatial heterogeneity of EMC microstructures in engineered tissues. This demonstrates the feasibility of the proposed method that nano- and micrometer scale alteration of the ECM structure can be detected and visualized at a whole-field level. Thus, we envision that this spectroscopic imaging approach could potentially serve as an effective characterization tool to nondestructively, accurately

  4. Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures (United States)

    Wohlleben, Wendel; Kolle, Susanne N.; Hasenkamp, Laura-Carolin; Böser, Alexander; Vogel, Sandra; von Vacano, Bernhard; van Ravenzwaay, Ben; Landsiedel, Robert


    We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

  5. Tissue culture and generation of autotetraploid plants of Sophora flavescens Aiton

    Directory of Open Access Journals (Sweden)

    Wei Kun-Hua


    Full Text Available Background: Sophora flavescens Aiton is an important medicinal plant in China. Early in vitro researches of S. flavescens were focused on callus induction and cell suspension culture, only a few were concerned with in vitro multiplication. Objective: To establish and optimize the rapid propagation technology of S. flavescens and to generate and characterize polyploid plants of S. flavescens. Materials and Methods: The different concentrations of 6-benzylaminopurine (BAP, indole-3-acetic acid (IAA and kinetin (KT were used to establish and screen the optimal rapid propagation technology of S. flavescens by orthogonal test; 0.2% colchicine solution was used to induce polyploid plants and the induced buds were identified by root-tip chromosome determination and stomatal apparatus observation. Results: A large number of buds could be induced directly from epicotyl and hypocotyl explants on the Murashige and Skoog medium (MS; 1962 supplemented with 1.4-1.6 mg/l 6-benzylaminopurine (BAP and 0.3 mg/l indole-3-acetic acid (IAA. More than 50 lines of autotetraploid plants were obtained. The chromosome number of the autotetraploid plantlet was 2n = 4x = 36. All tetraploid plants showed typical polyploid characteristics. Conclusion: Obtained autotetraploid lines will be of important genetic and breeding value and can be used for further selection and plant breeding.


    Directory of Open Access Journals (Sweden)

    Dolgikh Yu.I.


    Full Text Available The genetic variability induced by in vitro conditions known as somaclonal variation is of practical interest due to its potential uses in plant breeding but, on the other hand, if clonal propagation or transformation is main goal, it becomes an unwelcome phenomenon. Thus, it is important to know frequency, the genomic distribution, the mechanisms and factors influencing somaclonal variation. We studied variability of PCR-based DNA markers of cultured tissues and regenerated plants of maize and bread wheat. The original A188 line of maize and the somaclones obtained were tested using 38 RAPD and 10 ISSR primers. None of the A188 plants showed variation in the RAPD and ISSR spectra for any of the primers used. However, the PCR spectra obtained from the somaclones demonstrated some variations, i.e., 22 RAPD primers and 6 ISSR primers differentiated at least one somaclonal variant from the progenitor line. Six SCAR markers were developed based on several RAPD and ISSR fragments. The inheritance of these SCAR markers was verified in the selfing progeny of each somaclone in the R1–R4 generations and in the hybrids, with A188 as the parental line in the F1 and F2 generations. These markers were sequenced and bioinformatic searches were performed to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat transposon, precise miniature inverted repeat transposable element (MITE excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. In two groups of independently produced somaclones, the same features (morphological, molecular were variable, which confirms the theory of ‘hot spots’ occurring in the genome. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases

  7. Linear and nonlinear characterization of microbubbles and tissue using the Nakagami statistical model. (United States)

    Bahbah, N; Novell, A; Bouakaz, A; Djelouah, H


    The goal of this work is to exploit the statistical signatures for discrimination between biological tissues and contrast microbubbles in order to develop new strategies for contrast imaging and tissue characterization. For this purpose, the efficiency of the Nakagami statistical model, for describing the ultrasonic echoes of both contrast microbubbles and tissues, was investigated. Experimental measurements have been performed using a linear array probe connected to an open research platform. A commercially available in vitro phantom was used to mimic biological tissue in which SonoVue contrast microbubbles were flowing. Experimental ultrasound echoes have been filtered around the transmitted frequency (fundamental at 2.5MHz) and around twice the transmitted frequency (at 5MHz) for 2nd harmonic analysis, and a logarithmic compression was applied. The signals have been analyzed in order to evaluate the Nakagami parameter m, the scaling parameter Ω and the probability density function at both frequencies. Parametric images based on the Nakagami parameters map (Nakagami-mode images) were reconstructed and compared to B-mode images. Contrary to the B-mode image which is influenced by the system settings and user operations, the Nakagami parametric image is only based on the backscattered statistics of the ultrasonic signals in a local phantom. Such an imaging principle allows the Nakagami image to quantify the local scatterer concentrations in the phantom and to extract the backscattering information from the regions of the weakest echoes that may be lost in the conventional B-mode image. Results show that the tissue and microbubbles characterization is more sensitive in the 2nd harmonic mode when a logarithmic transform is used. These results would be useful for improving the ultrasound image quality and contrast detection in nonlinear mode.

  8. Somaclonal variation in tissue culture of banana cv. 'Kluai Num Wa'[Musa (ABB group

    Directory of Open Access Journals (Sweden)

    Te-chato, S.


    Full Text Available Two types of cytokinins, 6-Benzyladenine (BA and/or coconut water (CW and pH of medium were studied for their effects on shoot formation, growth and somaclonal variation of banana tissue culture. Thecultures were carried out on Murashige and Skoog (MS medium for 30 days. The average shoot length (5.82 cm was obtained in liquid medium supplemented with 15% CW. While a high number of shoots at 3.8shoots/explant were obtained in liquid medium supplemented with 5 mg/l BA. For fresh weight, liquid medium supplemented with 5 mg/l BA in combination with 15% CW gave the best results (2.26 g/shoot. Anoptimum pH for promotion shoot length (5.67 cm was 5.6 whereas the lower value (pH 4 promoted a high number of shoot formation (3.89 shoots/explant. The highest fresh weight of 1.33 g/shoot was obtained onmedium adjusted pH to 8. After maintaining the shoots by successive subculturing (3-4 week-intervals on MS medium with 5 mg/l BA several morphological abnormalities were obtained. Among those, chloroticleaves (1.5% were firstly observed in the first subculture and increased to 3% in the second subculture. Moreover, a narrow leaf at 25.76% was also observed in this period. Further subculture, more somaclonalvariation, such as a thin long shoots, nodular shoots and bamboo-like leaf, appeared. Isozyme marker revealed a difference in zymogram patterns among those somaclonal variants.

  9. [Rabies Tissue Culture Infection Test as an Alternative for the Mouse Inoculation Test (United States)

    Zanoni, R.; Hörnlimann, B.; Wandeler, A. I.; Kappeler, A.; Kipfer, R.; Peterhans, E.


    Rabies has disappeared from large parts of Switzerland. Due to systematic oral fox-vaccination campaings that started in 1987, cases of rabies in wild and domestic animals have been confined to the western frontier with France in the last three years. Nevertheless, some cases of severe exposition of man by rabid or rabies-suspect animals still occur. Rabies can be diagnosed in brain smears of infected animals with high specificity and sensitivity by a direct immunofluorescence method. According to WHO recommendations, negative results are to be confirmed in cases of a human exposition by intracerebral inoculation of brain suspensions in three-weeks-old mice. This method has an excellent sensitivity and is able to detect false-negative results in immunofluorescence, which occur in a very small percentage (0.043%). The disadvantage of this confirmatory assay is the sacrification of relatively high numbers of mice (in the Swiss rabies center about 1,300 animals each year), and the long time required for a final diagnosis: 7-20 days in positive, 21 days in negative cases. The cultivation of virus from brain suspensions on a mouse neuroblastoma cell line is a tempting alternative to the mouse inoculation test. This method usually provides a conclusive diagnosis within a few days. However, in our hands it showed in preliminary experiments an unsatisfactory sensitivity (80.7%). The necessity to carry out strict reproducibility controls in this assay has to be emphasized. Further work must be invested in the improvement of the rabies tissue culture infection test and a careful long-term comparison with the mouse inoculation test will be necessary before the mouse inoculation test can be replaced.

  10. A tissue culture technique for Amelanchier alnifolia%桤叶唐棣组培技术

    Institute of Scientific and Technical Information of China (English)

    王占龙; 黄立华; 龙忠伟


    A research on tissue culture of Amelanchier alnifolia was summarized and the optimal mediums were proposed in this paper.The medium of MS + BA 0.6 mg/L + NAA 0.12 mg/L + IBA 0.4 mg/L + sucrose 35 g/L + agar 8 g/L was optimal for inducement,the inducement rate could be up to 74% ; The medium of MS + BA 1.5 mg/L + IBA 1.0 mg/L + sucrose 35 g/L + agar 8 g/L was optimal for subculture,and the number of proliferation was 5.56 ; The medium of 1/2MS +NAA0.05 mg/L + IBA 1.0 mg/L + sucrose 35 g/L + agar 8 g/L was optimal for rooting,and the rooting rate was 91%.%对桤叶唐棣进行组织培养研究,结果表明,诱导培养基配方为MS+ BA 0.6 mg/L+ NAA 0.12 mg/L+ IBA0.4 mg/L+蔗糖35 g/L+琼脂8g/L,诱导率达到了74%;增殖培养基配方为MS+ BA 1.5 mg/L+ IBA 1.0 mg/L+蔗糖35 g/L+琼脂8 g/L,增殖倍数达到5.56倍;生根培养基配方为1/2MS+ NAA 0.05 mg/L+ IBA 1.0 mg/L+蔗糖35 g/L+琼脂8 g/L,生根率达91%.

  11. An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

    Directory of Open Access Journals (Sweden)

    Olga Krupkova


    Full Text Available Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP. Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1–20 U/mL or by the diffusion of interleukin-1 beta (IL-1β and tumor necrosis factor alpha (TNF-α (both 100 ng/mL from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS, prostaglandin-endoperoxide synthase 2 (PTGS2 and matrix metalloproteinases (MMP1, MMP3, and MMP13. The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.

  12. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines (United States)

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo


    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  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. A comprehensive characterization of cell cultures and xenografts derived from a human verrucous penile carcinoma

    DEFF Research Database (Denmark)

    Muñoz, Juan J; Drigo, Sandra A; Kuasne, Hellen;


    , and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix...... xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways......This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated...

  15. Growth of plant tissue cultures in simulated lunar soil: Implications for a lunar base Controlled Ecological Life Support System (CELSS) (United States)

    Venketeswaran, S.


    Experiments to determine whether plant tissue cultures can be grown in the presence of simulated lunar soil (SLS) and the effect of simulated lunar soil on the growth and morphogenesis of such cultures, as well as the effect upon the germination of seeds and the development of seedlings were carried out . Preliminary results on seed germination and seedling growth of rice and calli growth of winged bean and soybean indicate that there is no toxicity or inhibition caused by SLS. SLS can be used as a support medium with supplements of certain major and micro elements.

  16. Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system. (United States)

    Wewering, Franziska; Jouy, Florent; Wissenbach, Dirk K; Gebauer, Scarlett; Blüher, Matthias; Gebhardt, Rolf; Pirow, Ralph; von Bergen, Martin; Kalkhof, Stefan; Luch, Andreas; Zellmer, Sebastian


    Liver injury as a result of a sterile inflammation is closely linked to the activation of immune cells, including macrophages, by damaged hepatocytes. This interaction between immune cells and hepatocytes is as yet not considered in any of the in vitro test systems applied during the generation of new drugs. Here, we established and characterized a novel in vitro co-culture model with two human cell lines, HepG2 and differentiated THP-1. Ketoconazole, an antifungal drug known for its hepatotoxicity, was used as a model compound in the testing of the co-culture. Single cultures of HepG2 and THP-1 cells were studied as controls. Different metabolism patterns of ketoconazole were observed for the single and co-culture incubations as well as for the different cell types. The main metabolite N-deacetyl ketoconazole was found in cell pellets, but not in supernatants of cell cultures. Global proteome analysis showed that the NRF2-mediated stress response and the CXCL8 (IL-8) pathway were induced by ketoconazole treatment under co-culture conditions. The upregulation and ketoconazole-induced secretion of several pro-inflammatory cytokines, including CXCL8, TNF-α and CCL3, was observed in the co-culture system only, but not in single cell cultures. Taking together, we provide evidence that the co-culture model applied might be suitable to serve as tool for the prediction of chemical-induced sterile inflammation in liver tissue in vivo.

  17. Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

    Energy Technology Data Exchange (ETDEWEB)

    Gautam, Sneh [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, Roorkee, 247667 (India); Dinda, Amit Kumar [Department of Pathology, All India Institute of Medical Science, New Delhi, 110029 (India); Mishra, Narayan Chandra, E-mail: [Department of Polymer and Process Engineering, Indian Institute of Technology Roorkee, Roorkee, 247667 (India)


    In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications. - Highlights: ► PCL/Gelatin scaffold was successfully fabricated by electrospinning method. ► PCL in CHCl{sub 3}/CH{sub 3}OH and gelatin in acetic acid: a novel polymer-solvent system. ► The morphology of nanofibers was influenced by the weight ratio of PCL/gelatin. ► Chemical interactions between PCL and gelatin molecules enhanced cell growth. ► Cell culture studies indicate the suitability of scaffold for tissue regeneration.

  18. Ectopic Thyroid Tissue in the Mediastinum Characterized by Histology and Functional Imaging with I-123 SPECT/CT (United States)

    Hummel, Jed; Wachsmann, Jason; Carrick, Kelley; Oz, Orhan K.; Mathews, Dana


    Ectopic thyroid tissue is a rare entity and when discovered it is typically along the pathway of embryologic migration of the thyroid. We present a case of incidental finding of ectopic thyroid tissue within mediastinum in a 61-year-old female patient with a history of total thyroidectomy for thyroiditis and nodules. The patient presented to emergency room with cough and right chest pain and underwent a chest computed tomographic angiogram (CTA) to exclude pulmonary embolism as part of chest pain workup. One right paratracheal mediastinal soft tissue nodule was visualized on the images of CTA. This right paratracheal soft tissue mass was found to be ectopic benign thyroid tissue by histological analysis of the biopsied tissue samples. The function of this ectopic thyroid tissue was characterized by I-123 radioiodine uptake and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. This case illustrates that ectopic thyroid tissue should be included for differential diagnosis of a hyperdense soft tissue mass located within mediastinum. I-123 SPECT/CT is useful for guiding tissue biopsy of ectopic thyroid tissue distant from orthotopic thyroid gland and functional and anatomic characterization of mediastinal ectopic thyroid tissue for surgical resection when it is medically necessary. PMID:28251012

  19. Ectopic Thyroid Tissue in the Mediastinum Characterized by Histology and Functional Imaging with I-123 SPECT/CT. (United States)

    Hummel, Jed; Wachsmann, Jason; Carrick, Kelley; Oz, Orhan K; Mathews, Dana; Peng, Fangyu


    Ectopic thyroid tissue is a rare entity and when discovered it is typically along the pathway of embryologic migration of the thyroid. We present a case of incidental finding of ectopic thyroid tissue within mediastinum in a 61-year-old female patient with a history of total thyroidectomy for thyroiditis and nodules. The patient presented to emergency room with cough and right chest pain and underwent a chest computed tomographic angiogram (CTA) to exclude pulmonary embolism as part of chest pain workup. One right paratracheal mediastinal soft tissue nodule was visualized on the images of CTA. This right paratracheal soft tissue mass was found to be ectopic benign thyroid tissue by histological analysis of the biopsied tissue samples. The function of this ectopic thyroid tissue was characterized by I-123 radioiodine uptake and single photon emission computed tomography/computed tomography (SPECT/CT) imaging. This case illustrates that ectopic thyroid tissue should be included for differential diagnosis of a hyperdense soft tissue mass located within mediastinum. I-123 SPECT/CT is useful for guiding tissue biopsy of ectopic thyroid tissue distant from orthotopic thyroid gland and functional and anatomic characterization of mediastinal ectopic thyroid tissue for surgical resection when it is medically necessary.

  20. Ectopic Thyroid Tissue in the Mediastinum Characterized by Histology and Functional Imaging with I-123 SPECT/CT

    Directory of Open Access Journals (Sweden)

    Jed Hummel


    Full Text Available Ectopic thyroid tissue is a rare entity and when discovered it is typically along the pathway of embryologic migration of the thyroid. We present a case of incidental finding of ectopic thyroid tissue within mediastinum in a 61-year-old female patient with a history of total thyroidectomy for thyroiditis and nodules. The patient presented to emergency room with cough and right chest pain and underwent a chest computed tomographic angiogram (CTA to exclude pulmonary embolism as part of chest pain workup. One right paratracheal mediastinal soft tissue nodule was visualized on the images of CTA. This right paratracheal soft tissue mass was found to be ectopic benign thyroid tissue by histological analysis of the biopsied tissue samples. The function of this ectopic thyroid tissue was characterized by I-123 radioiodine uptake and single photon emission computed tomography/computed tomography (SPECT/CT imaging. This case illustrates that ectopic thyroid tissue should be included for differential diagnosis of a hyperdense soft tissue mass located within mediastinum. I-123 SPECT/CT is useful for guiding tissue biopsy of ectopic thyroid tissue distant from orthotopic thyroid gland and functional and anatomic characterization of mediastinal ectopic thyroid tissue for surgical resection when it is medically necessary.

  1. Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies. (United States)

    Melgarejo-Ramírez, Y; Sánchez-Sánchez, R; García-López, J; Brena-Molina, A M; Gutiérrez-Gómez, C; Ibarra, C; Velasquillo, C


    The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

  2. Advances in Adipose-Derived Stem Cells Isolation, Characterization, and Application in Regenerative Tissue Engineering. (United States)

    Wankhade, Umesh D; Shen, Michael; Kolhe, Ravindra; Fulzele, Sadanand


    Obesity is a complex, multifactorial disease that has been extensively researched in recent times. Obesity is characterized by excess deposition of adipose tissue in response to surplus energy. Despite the negative connotations of adipose tissue (AT), it serves as a critical endocrine organ. Adipose tissue is a source of several adipokines and cytokines which have been deemed important for both normal metabolic function and disease formation. The discoveries of metabolically active brown AT in adult humans and adipose tissue derived stem cells (ADSC) have been key findings in the past decade with potential therapeutic implications. ADSCs represent an enticing pool of multipotent adult stem cells because of their noncontroversial nature, relative abundance, ease of isolation, and expandability. A decade and a half since the discovery of ADSCs, the scientific community is still working to uncover their therapeutic potential in a wide range of diseases. In this review, we provide an overview of the recent developments in the field of ADSCs and examine their potential use in transplantation and cell-based therapies for the regeneration of diseased organs and systems. We also hope to provide perspective on how to best utilize this readily available, powerful pool of stem cells in the future.

  3. Identification and characterization of tissues by T2-selective whole-body proton NMR imaging. (United States)

    Gersonde, K; Tolxdorff, T; Felsberg, L


    T2-selective proton imaging at 10 MHz is based on a Carr-Purcell-Meiboom-Gill pulse sequence with at least 36 echoes and 2 tau greater than or equal to 6 ms employing the projection reconstruction technique. The transverse magnetization decay of the volume elements is multiexponential composed of up to three monoexponential T2 components. The T2 values are arranged in T2 histograms which are slice specific. T2 windows are defined in the T2 histogram and used for imaging and for selecting T2 values which are assigned to proton classes and tissues. Tissue characterization is based on the visualization of differences in T2 behavior. The T2 values are transformed into 256 gray levels or, by use of a red-green-blue lookup table, into a rainbow code. Two display techniques have been developed and implemented in the RWTH Aachen Magnetic Resonance Software System (RAMSES): (i) for nonselective T2 imaging with secondary T2 selection and encoding of molecular classes on the basis of T2 values; (ii) for T2-selective imaging by employing combined primary and secondary T2 selection and encoding particular tissues and variations therein. The tissues can be identified by selecting different classes of molecules, like water, lipids, and proteins, when shifting the primary T2 window on the T2 axis of the T2 histogram.

  4. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

    NARCIS (Netherlands)

    Booij, T.H.; Klop, M.J.; Yan, K.; Szántai-Kis, C.; Szokol, B.; Orfi, L .; Water, van de B.; Keri, G.; Price, L.S.


    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses

  5. Preparation and characterization of aloe vera blended collagen-chitosan composite scaffold for tissue engineering applications. (United States)

    Jithendra, Panneerselvam; Rajam, Abraham Merlin; Kalaivani, Thambiran; Mandal, Asit Baran; Rose, Chellan


    Collagen-Chitosan (COL-CS) scaffolds supplemented with different concentrations (0.1-0.5%) of aloe vera (AV) were prepared and tested in vitro for their possible application in tissue engineering. After studying the microstructure and mechanical properties of all the composite preparations, a 0.2% AV blended COL-CS scaffold was chosen for further studies. Scaffolds were examined by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and thermogravimetry analysis (TGA) to understand the intermolecular interactions and their influence on the thermal property of the complex composite. Swelling property in phosphate buffered saline (pH 7.4) and in vitro biodegradability by collagenase digestion method were monitored to assess the stability of the scaffold in a physiological medium in a hydrated condition, and to assay its resistance against enzymatic forces. The scanning electron microscope (SEM) image of the scaffold samples showed porous architecture with gradual change in their morphology and reduced tensile properties with increasing aloe vera concentration. The FTIR spectrum revealed the overlap of the AV absorption peak with the absorption peak of COL-CS. The inclusion of AV to COL-CS increased the thermal stability as well as hydrophilicity of the scaffolds. Cell culture studies on the scaffold showed enhanced growth and proliferation of fibroblasts (3T3L1) without exhibiting any toxicity. Also, normal cell morphology and proliferation were observed by fluorescence microscopy and SEM. The rate of cell growth in the presence/absence of aloe vera in the scaffolds was in the order: COL-CS-AV > COL-CS > TCP (tissue culture polystyrene plate). These results suggested that the aloe vera gel-blended COL-CS scaffolds could be a promising candidate for tissue engineering applications.

  6. Characterization of microbes in prosthetic joint specimens by culture-independent molecular methods

    DEFF Research Database (Denmark)

    Xu, Yijuan; Rudkjøbing, Vibeke Børsholt; Simonsen, Ole

    was to investigate the microbial diversity in surgical samples (eg. synovial fluid, periprosthetic tissue, removed prosthesis) from 22 prosthetic patients using a range of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, phylogeny, quantitative PCR (qPCR), and fluorescence...... in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Overall, the results of culture-based and molecular methods showed concordant results for 13 patients and discrepant results for 6 patients. In the remaining cases, culture methods identified one species or a group...... approach, and FISH and confocal scanning laser microscopy visualized the presence of both single cells and microcolonies. In conclusion, cultureindependent methods identified more species and more polymicrobial infections than standard culturebased methods. Little is presently known about the pathogenesis...

  7. Tissue characterization of skin ulcer for bacterial infection by multiple statistical analysis of echo amplitude envelope (United States)

    Omura, Masaaki; Yoshida, Kenji; Kohta, Masushi; Kubo, Takabumi; Ishiguro, Toshimichi; Kobayashi, Kazuto; Hozumi, Naohiro; Yamaguchi, Tadashi


    To characterize skin ulcers for bacterial infection, quantitative ultrasound (QUS) parameters were estimated by the multiple statistical analysis of the echo amplitude envelope based on both Weibull and generalized gamma distributions and the ratio of mean to standard deviation of the echo amplitude envelope. Measurement objects were three rat models (noninfection, critical colonization, and infection models). Ultrasound data were acquired using a modified ultrasonic diagnosis system with a center frequency of 11 MHz. In parallel, histopathological images and two-dimensional map of speed of sound (SoS) were observed. It was possible to detect typical tissue characteristics such as infection by focusing on the relationship of QUS parameters and to indicate the characteristic differences that were consistent with the scatterer structure. Additionally, the histopathological characteristics and SoS of noninfected and infected tissues were matched to the characteristics of QUS parameters in each rat model.

  8. A Case Study of Native Plants at Industrially Contaminated Area in Bhopal and Their Tissue Culture Approach for Phytoremediation


    Ashwini A Waoo; Sujata Ganguly; Swati Khare


    In phytoremediation techniques,experiments were done with normal soil-grown or hydroponically grown plants. In recent days, more and more effort is directed toward research to understand and improve the performance of plants in remediation technologies, the number of results obtained with the help of in vitro plant cell and tissue cultures is rapidly increasing.This review focuses on the report of various phytoremediation technologies, paying special attention to removal of pollutants from so...

  9. Tissue-culture light sheet fluorescence microscopy (TC-LSFM) allows long-term imaging of three-dimensional cell cultures under controlled conditions. (United States)

    Pampaloni, Francesco; Berge, Ulrich; Marmaras, Anastasios; Horvath, Peter; Kroschewski, Ruth; Stelzer, Ernst H K


    Fluorescence long-term imaging of cellular processes in three-dimensional cultures requires the control of media supply, temperature, and pH, as well as minimal photodamage. We describe a system based on a light sheet fluorescence microscope (LSFM), which is optimized for long-term, multi-position imaging of three-dimensional in-gel cell cultures. The system integrates a stable culture condition control system in the optical path of the light-sheet microscope. A further essential element is a biocompatible agarose container suitable for the LSFM, in which any cell type can be cultured in different gel matrices. The TC-LSFM allows studying any in vitro cultured cell type reacting to, dividing in, or migrating through a three-dimensional extracellular matrix (ECM) gel. For this reason we called it "tissue culture-LSFM" (TC-LSFM). The TC-LSFM system allows fast imaging at multiple locations within a millimeter-sized ECM gel. This increases the number of analyzed events and allows testing population effects. As an example, we show the maturation of a cyst of MDCK (canine kidney epithelial) cells over a period of three days. Moreover, we imaged, tracked, and analyzed MDCK cells during the first five days of cell aggregate formation and discovered a remarkable heterogeneity in cell cycle lengths and an interesting cell death pattern. Thus, TC-LSFM allows performing new long-term assays assessing cellular behavior in three-dimensional ECM-gel cultures. For example migration, invasion or differentiation in epithelial cell systems, stem cells, as well as cancer cells can be investigated.

  10. A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture.

    Directory of Open Access Journals (Sweden)

    Nirmalya Chatterjee

    Full Text Available This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences, reporter genes (eGFP, or DsRed.T4, and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase.

  11. Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

    Directory of Open Access Journals (Sweden)

    Saburo Hidaka


    Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  12. Production and characterization of chitosan/gelatin/β-TCP scaffolds for improved bone tissue regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Serra, I.R.; Fradique, R.; Vallejo, M.C.S.; Correia, T.R.; Miguel, S.P.; Correia, I.J., E-mail:


    Recently, bone tissue engineering emerged as a viable therapeutic alternative, comprising bone implants and new personalized scaffolds to be used in bone replacement and regeneration. In this study, biocompatible scaffolds were produced by freeze-drying, using different formulations (chitosan, chitosan/gelatin, chitosan/β-TCP and chitosan/gelatin/β-TCP) to be used as temporary templates during bone tissue regeneration. Sample characterization was performed through attenuated total reflectance-Fourier transform infrared spectroscopy, X-ray diffraction and energy dispersive spectroscopy analysis. Mechanical characterization and porosity analysis were performed through uniaxial compression test and liquid displacement method, respectively. In vitro studies were also done to evaluate the biomineralization activity and the cytotoxic profile of the scaffolds. Scanning electron and confocal microscopy analysis were used to study cell adhesion and proliferation at the scaffold surface and within their structure. Moreover, the antibacterial activity of the scaffolds was also evaluated through the agar diffusion method. Overall, the results obtained revealed that the produced scaffolds are bioactive and biocompatible, allow cell internalization and show antimicrobial activity against Staphylococcus aureus. Such, make these 3D structures as potential candidates for being used on the bone tissue regeneration, since they promote cell adhesion and proliferation and also prevent biofilm development at their surfaces, which is usually the main cause of implant failure. - Highlights: • Production of 3D scaffolds composed by chitosan/gelatin/β-TCP by freeze-drying for bone regeneration • Physicochemical characterization of the bone substitutes by SEM, FTIR, XRD and EDS • Evaluation of the cytotoxic profile and antibacterial activity of the 3D structures through in vitro assays.

  13. Tissue Culture of Gerbera jamesonii%非洲菊的组织培养研究

    Institute of Scientific and Technical Information of China (English)

    杨尧; 任雪; 杜兴翠; 章四庆; 郭巧会; 赖齐贤


    为提供非洲菊化学诱变的基础材料以及相应的组培操作技术,采用非洲菊品种‘琳达’的幼嫩花托为外植体进行组织培养研究,外植体最佳消毒时间为流水冲洗1 h,75%的酒精浸泡30 s,0.1%的HgCl2灭菌12 min;诱导产生愈伤组织的最适培养基为:MS+6-BA 5.0 mg/L+NAA 0.5 mg/L+蔗糖3%(w/v)+琼脂粉7%(w/v);增殖培养基配方MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+蔗糖3%(w/v)+琼脂粉7%(w/v)为最适配方;生根壮苗培养基以1/2MS+NAA0.1 mg/L+蔗糖2%+琼脂粉7%为最佳。试验显示以花托为外植体较为容易进行表面灭菌,诱导愈伤组织并分化丛生芽,从而快速建立离体快繁体系。%To provide the basis of the materials for Gerbera’s chemical inducement;and the corresponding set of technique.Using gerbera varieties young receptacle of‘Linda’as explants to tissue culture.the best sterilization time on explants were washed for one hour and sterilized with 75% ethyl alcohol for 30 s,0.1% HgCl2 for 12 minutes,the calluses introduction medium for in vitro culture was MS(murashige and skoog) with 6-BA(5.0 mg/L) + NAA(0.5 mg/L) + sucrose(3%) + agar(7%),the multiplication medium was MS with 6-BA(1.0 mg/L)+NAA(0.1mg/L) + sucrose(3%)+agar(7%),the rooting medium was 1/2MS with NAA(0.1 mg/L)+ sucrose(2%)+agar(7%).From the experiments,it was evident that young receptacle as explants was more easy to surface sterilization,callus induction and multiple shoot clumps,then established rapid propagation system of Gerbera.

  14. Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue

    Directory of Open Access Journals (Sweden)

    S. Austin eHammond


    Full Text Available Nanoparticles (NPs, materials that have one dimension less than 100 nm, are used in manufacturing, health and food products, and consumer products including cosmetics, clothing and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs, thyroxine and 3,3’,5-triiodothyronine (T3, are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs. We used the cultured tailfin (C-fin assay to examine the exposure effects of 0.1-10 nM (~8-800 ng/L of three types of ~20 nm TiO2 NPs (P25, M212, M262 and micron-sized TiO2 (μTiO2 ±10 nM T3. The actual Ti levels were 40.9 – 64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR was used to measure the relative amounts of mRNA transcripts encoding TH-responsive thyroid hormone receptors (thra and thrb and Rana larval keratin type I (rlk1, as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30, superoxide dismutase (sod, and catalase (cat. The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25 and M212 while no effect was observed with M262 exposure. Therefore the risk of adversely affecting amphibian tissue by disrupting TH-signalling or inducing cellular stress is low for these compounds relative to other previously

  15. Tissue Culture of Begonia f imbristipula%降压草的组织培养技术研究

    Institute of Scientific and Technical Information of China (English)

    郭福生; 陈淑华; 王漫; 叶雅玲


    Taking Begonia fimbristipula‘Erhao’ which was cultivated by Baicheng Academy of Forestry as experi‐mental materials ,tissue culture of stem with buds of Begonia fimbristipula was conducted .Tissue culture program and culture medium formula were selected .Result shows that MS+6-BA 0 .5 mg · L -1 +NAA 0 .1 mg · L -1 is the suitable culture medium for inducing bud;MS+6-BA 1 .0 mg · L -1 +NAA 0 .5 mg · L -1 +IAA 0 .1 mg · L -1 is the suitable proliferation medium .%以白城市林业科学研究院选育的新品种降压草2号为试材,利用其带芽茎段进行组织培养,筛选降压草组织培养程序及培养基配方。结果表明:适宜的腋芽诱导培养基为MS+6-BA0.5 mg 灋L -1+ NAA0.1 mg 灋L -1,适宜的增殖培养基为MS+6-BA1.0 mg 灋L -1+ NAA0.5 mg 灋L -1+IAA0.1 mg 灋L -1。

  16. Characterization and Differentiation of Stem Cells Isolated from Human Newborn Foreskin Tissue. (United States)

    Somuncu, Özge Sezin; Taşlı, Pakize Neslihan; Şişli, Hatice Burcu; Somuncu, Salih; Şahin, Fikrettin


    Circumcision is described as a cultural, medical, and religious process which states surgical removal of the foreskin either partly or fully. Cells isolated from the circumcised tissues are referred as foreskin cells. They have been thought as feeder cell lines for embryonic stem cells. Their fibroblastic properties were also utilized for several experiments. The waste tissues that remain after the circumcision thought to have stem cell properties. Therefore, there have been very few attempts to expose their stem cell properties without turning them into induced pluripotent stem cells. Although stem cell isolation from prepuce and their mesenchymal multilineage differentiation potential have been presented many times in the literature, the current study explored hematopoietical phenotype of newborn foreskin stem cells for the first time. According to the results, human newborn foreskin stem cells (hnFSSCs) were identified by their capability to turn into all three germ layer cell types under in vitro conditions. In addition, these cells have exhibited a stable phenotype and have remained as a monolayer in vitro. hnFSSCs suggested to carry different treatment potentials for bone damages, cartilage problems, nerve damages, lesion formations, and other diseases that are derive from mesodermal, endodermal, and ectodermal origins. Owing to the location of the tissue in the body and differentiation capabilities of hnFSSCs, these cells can be considered as easily obtainable and utilizable even better than the other stem cell sources. In addition, hnFSSCs offers a great potential for tissue engineering approaches due to exhibiting embryonic stem cell-like characteristics, not having any ethical issues, and teratoma induction as in embryonic stem cell applications.

  17. Factors affecting proliferation and elongation of shoots of Phak Liang (Gnetum gnemon Linn. through tissue culture technique

    Directory of Open Access Journals (Sweden)

    Te-chato, S.


    Full Text Available The tissue culture of Phak Liang (Gnetum gnemon Linn. was investigated for micropropagation. The types of explant, culture media, types and concentrations of plant growth regulators, orientation of explant and section of explant were tested for their efficacy in inducing and proliferating shoot buds. The elongation of shoots and root induction was also studied. Young leaves gave the highest number of shoot buds when they were cultured in Murashige and Skoog (MS medium supplemented with 0.25 mg/l IBA and 1.53 mg/l BA. The medium supplemented with 0.25 mg/l thidiazuron (TDZ alone provided the best result on multiple shoot bud induction both in percentage of explant forming shoots and number of shoot buds per explant. The percentage of explant forming shoot buds and number of shoot buds obtained from leaves were 90% and 26.50 shoot buds, while those from stems were 96.25% and 23.00 shoot buds, respectively. One hundred percent friable callus was induced from stem explant in the same medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D after 2 months of culture. Culturing whole leaf in the position of dorsal contact with medium gave the best multiple shoot bud formation of 92% and 23.00 shoot buds/explant. Cutting stem into half and culturing in horizontal position gave the best multiple shoot bud formation of 96% and 23.00 shoot buds/explant after culture for 2 months. The best elongation of shoot buds (2.54 shoots derived from cultured leaves was induced in the liquid medium. While stem-derived shoot buds (3.45 shoots was induced in the solid medium of the same medium components. However, root could not be induced from elongated shoots.

  18. Small-window parametric imaging based on information entropy for ultrasound tissue characterization (United States)

    Tsui, Po-Hsiang; Chen, Chin-Kuo; Kuo, Wen-Hung; Chang, King-Jen; Fang, Jui; Ma, Hsiang-Yang; Chou, Dean


    Constructing ultrasound statistical parametric images by using a sliding window is a widely adopted strategy for characterizing tissues. Deficiency in spatial resolution, the appearance of boundary artifacts, and the prerequisite data distribution limit the practicability of statistical parametric imaging. In this study, small-window entropy parametric imaging was proposed to overcome the above problems. Simulations and measurements of phantoms were executed to acquire backscattered radiofrequency (RF) signals, which were processed to explore the feasibility of small-window entropy imaging in detecting scatterer properties. To validate the ability of entropy imaging in tissue characterization, measurements of benign and malignant breast tumors were conducted (n = 63) to compare performances of conventional statistical parametric (based on Nakagami distribution) and entropy imaging by the receiver operating characteristic (ROC) curve analysis. The simulation and phantom results revealed that entropy images constructed using a small sliding window (side length = 1 pulse length) adequately describe changes in scatterer properties. The area under the ROC for using small-window entropy imaging to classify tumors was 0.89, which was higher than 0.79 obtained using statistical parametric imaging. In particular, boundary artifacts were largely suppressed in the proposed imaging technique. Entropy enables using a small window for implementing ultrasound parametric imaging. PMID:28106118

  19. Dry-cured ham tissue characterization by fast field cycling NMR relaxometry and quantitative magnetization transfer. (United States)

    Bajd, Franci; Gradišek, Anton; Apih, Tomaž; Serša, Igor


    Fast field cycling (FFC) and quantitative magnetization transfer (qMT) NMR methods are two powerful tools in NMR analysis of biological tissues. The qMT method is well established in biomedical NMR applications, while the FFC method is often used in investigations of molecular dynamics on which longitudinal NMR relaxation times of the investigated material critically depend. Despite their proven analytical potential, these two methods were rarely used in NMR studies of food, especially when combined together. In our study, we demonstrate the feasibility of a combined FFC/qMT-NMR approach for the fast and nondestructive characterization of dry-curing ham tissues differing by protein content. The characterization is based on quantifying the pure quadrupolar peak area (area under the quadrupolar contribution of dispersion curve obtained by FFC-NMR) and the restricted magnetization pool size (obtained by qMT-NMR). Both quantities correlate well with concentration of partially immobilized, nitrogen-containing and proton magnetization exchanging muscle proteins. Therefore, these two quantities could serve as potential markers for dry-curing process monitoring. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Mapping and characterization of iron compounds in Alzheimer's tissue

    Energy Technology Data Exchange (ETDEWEB)

    Collingwood, Joanna; Dobson, Jon [Keele


    Understanding the management of iron in the brain is of great importance in the study of neurodegeneration, where regional iron overload is frequently evident. A variety of approaches have been employed, from quantifying iron in various anatomical structures, to identifying genetic risk factors related to iron metabolism, and exploring chelation approaches to tackle iron overload in neurodegenerative disease. However, the ease with which iron can change valence state ensures that it is present in vivo in a wide variety of forms, both soluble and insoluble. Here, we review recent developments in approaches to locate and identify iron compounds in neurodegenerative tissue. In addition to complementary techniques that allow us to quantify and identify iron compounds using magnetometry, extraction, and electron microscopy, we are utilizing a powerful combined mapping/characterization approach with synchrotron X-rays. This has enabled the location and characterization of iron accumulations containing magnetite and ferritin in human Alzheimer's disease (AD) brain tissue sections in situ at micron-resolution. It is hoped that such approaches will contribute to our understanding of the role of unusual iron accumulations in disease pathogenesis, and optimise the potential to use brain iron as a clinical biomarker for early detection and diagnosis.

  1. Automated characterization of normal and pathologic lung tissue by topological texture analysis of multidetector CT (United States)

    Boehm, H. F.; Fink, C.; Becker, C.; Reiser, M.


    Reliable and accurate methods for objective quantitative assessment of parenchymal alterations in the lung are necessary for diagnosis, treatment and follow-up of pulmonary diseases. Two major types of alterations are pulmonary emphysema and fibrosis, emphysema being characterized by abnormal enlargement of the air spaces distal to the terminal, nonrespiratory bronchiole, accompanied by destructive changes of the alveolar walls. The main characteristic of fibrosis is coursening of the interstitial fibers and compaction of the pulmonary tissue. With the ability to display anatomy free from superimposing structures and greater visual clarity, Multi-Detector-CT has shown to be more sensitive than the chest radiograph in identifying alterations of lung parenchyma. In automated evaluation of pulmonary CT-scans, quantitative image processing techniques are applied for objective evaluation of the data. A number of methods have been proposed in the past, most of which utilize simple densitometric tissue features based on the mean X-ray attenuation coefficients expressed in terms of Hounsfield Units [HU]. Due to partial volume effects, most of the density-based methodologies tend to fail, namely in cases, where emphysema and fibrosis occur within narrow spatial limits. In this study, we propose a methodology based upon the topological assessment of graylevel distribution in the 3D image data of lung tissue which provides a way of improving quantitative CT evaluation. Results are compared to the more established density-based methods.

  2. Three isozymes of peptidylarginine deiminase in the chicken: molecular cloning, characterization, and tissue distribution. (United States)

    Shimizu, Akira; Handa, Kenji; Honda, Tomonori; Abe, Naoki; Kojima, Toshio; Takahara, Hidenari


    Peptidylarginine deiminase (PAD; EC is a post-translational modification enzyme that catalyzes the conversion of protein-bound arginine to citrulline (deimination) in a calcium ion dependent manner. Although PADI genes are widely conserved among vertebrates, their function in the chicken is poorly understood. Here, we cloned and sequenced three chicken PADI cDNAs and analyzed the expression of their proteins in various tissues. Immunoblotting analysis showed that chicken PAD1 and PAD3 were present in cells of several central neuron system tissues including the retina; the chicken PAD2 protein was not detected in any tissue. We expressed recombinant chicken PADs in insect cells and characterized their enzymatic properties. The chicken PAD1 and PAD3 recombinant proteins required calcium ions as an essential cofactor for their catalytic activity. The two recombinant proteins showed similar substrate specificities toward synthetic arginine derivatives. By contrast to them, chicken PAD2 did not show any activity. We found that one of the conserved active centers in mammalian PADs had been altered in chicken PAD2; we prepared a reverse mutant but we did not detect an activity. We conclude that chicken PAD1 and PAD3 might play specific roles in the nervous system, but that chicken PAD2 might not be functional under normal physiological conditions.

  3. In-situ Characterization and Mapping of Iron Compounds in Alzheimer's Tissue

    Energy Technology Data Exchange (ETDEWEB)

    Collingwood, J F; Mikhaylova, A; Davidson, M; Batich, C; Streit, W J; Terry, J; Dobson, J [IIT; (Keele); (Florida)


    There is a well-established link between iron overload in the brain and pathology associated with neurodegeneration in a variety of disorders such as Alzheimer's (AD), Parkinson's (PD) and Huntington's (HD) diseases. This association was first discovered in AD by Goodman in 1953, where, in addition to abnormally high concentrations of iron in autopsy brain tissue, iron has also been shown to accumulate at sites of brain pathology such as senile plaques. However, since this discovery, progress in understanding the origin, role and nature of iron compounds associated with neurodegeneration has been slow. Here we report, for the first time, the location and characterization of iron compounds in human AD brain tissue sections. Iron fluorescence was mapped over a frontal-lobe tissue section from an Alzheimer's patient, and anomalous iron concentrations were identified using synchrotron X-ray absorption techniques at 5 {micro}m spatial resolution. Concentrations of ferritin and magnetite, a magnetic iron oxide potentially indicating disrupted brain-iron metabolism, were evident. These results demonstrate a practical means of correlating iron compounds and disease pathology in-situ and have clear implications for disease pathogenesis and potential therapies.

  4. Extraction and characterization of candidate bioactive compounds in different tissues from salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Susan Skanderup Falkenberg


    Full Text Available Summary. There is an interest in bioprospecting organisms from the aquatic environment to find novel bioactive compounds with health promoting or other functional properties. The aim of this study was to evaluate extracts from  untreated and heat-treated salmon tissues for their radical scavenging activities and for their ability to inhibit activity of the proteases angiotensin I-converting enzyme (ACE and dipeptidyl peptidase 4 (DPP-4. In vitro assays were used to detect these activities and the corresponding candidate bioactive compounds were characterized by LC-MS/MS.Radical scavenging activity was detected in <10kDa extracts of gills, belly flap muscle and skin with EC50 values of 39, 82 and 100 µg/mL, respectively. No ACE or DPP-4 inhibiting activity could be detected. LC-MS/MS analysis of dominating compounds in active fractions from size exclusion chromatography showed that families of related compounds were found in several fractions from different tissues but most pronounced in gills. One family was defined according to content of a specific amino acid sequence (PW. Three families were defined by the m/z value of the smallest compound reported in each family (219, 434 and 403. The three latter families did not contain standard unmodified amino acids, indicating peptides with modified amino acids or other kinds of molecules.Industrial relevance. Bioprospecting in fish tissue traditionally regarded as waste can lead to detection of novel natural bioactive compounds including peptides, which could have nutritional, pharmaceutical or other functional value and be used in health and functional foods, thus increasing the value adding of secondary marine products. A number of  naturally occurring antimicrobial  peptides have been characterized from fish skin and gills, such as piscidins, but these and other fish tissues may contain numerous other compounds with bioactive properties. Such compounds could be extracted by the subsection of

  5. Rate of change in central corneal thickness: a viability indicator for conventional drainage tissues in organ culture. (United States)

    Wan, Z; Brigatti, L; Ranger-Moore, J; Ethier, C R; Stamer, W D


    Organ culture of human anterior segments is a powerful tool for understanding trabecular meshwork biology. However, data from a significant percentage of cultured anterior segments are unusable because tissues fail to meet quality control requirements, such as having adequate trabecular meshwork histology. The purpose of the present study was to evaluate a novel, real time method for assessing the viability of conventional drainage tissues in the human anterior segment perfusion model. Twenty-two human anterior segments were perfusion cultured using standard techniques for one week while measuring outflow facility and central corneal thickness (CCT). After perfusion-fixation, toludine blue-stained histological sections of drainage tissues from all four quadrants of each anterior segment were graded and endothelial cell nuclei from cornea centers were stained with 4',6-diamidino-2-phenylindole and counted. We found that most anterior segments with a stable outflow facility had a CCT that decreased over time, while anterior segments with an unstable outflow facility had CCT measurements that failed to decrease over time (P<0.01). When comparing CCT measurements to histological appearance of outflow tissues, we found that in 11/11 cases, anterior segments with an acceptable histological score had a negative CCT slope (P<0.01). Conversely in 3/4 instances, anterior segments with an unacceptable histological score had a positive CCT slope. Lastly, we observed a significant relationship between CCT measurements and corneal endothelial density (P<0.01). Thus, the simple procedure of measuring CCT during anterior segment perfusion provides a second useful measure to assess the viability of the anterior segment during the perfusion process.

  6. Characterization of the diffuse mucosal associated lymphoid tissue of feline small intestine. (United States)

    Roccabianca, P; Woo, J C; Moore, P F


    Characterization of the feline intestinal mucosal associated lymphoid tissue (MALT) will facilitate investigation of intestinal disease in the cat and promote the cat as an animal model for a range of human diseases which involve the intestinal lymphoid tissue. This includes inflammatory bowel disease, viral and non-viral associated intestinal lymphomas and immunodeficiency associated syndromes. Morphologic and phenotypic characterization of the normal small intestinal diffuse MALT in 22 SPF cats was performed using flow cytometry and cytology on isolated intestinal leukocytes from the intra-epithelial and lamina proprial compartments, as well as immunohistology on tissues from the feline duodenum, jejunum and ileum. The intra-epithelial compartment (IEC) was dominated by lymphocytes (>85%) which frequently contained intracytoplasmic granules. The most striking findings in the IEC were the elevated percentages of CD8 alpha+ lymphocytes (40%), presumed to express CD8 alpha alpha chains, and CD4-/CD8- (double negative) lymphocytes (44%), and the consistent presence of a minor subpopulation of CD3+/CD11d+ IELs (6%). Small percentages of CD4+ lymphocytes (10%) were observed such that the IEL CD4:CD8 ratio (0.25) was low. The LPC also contained a majority of T cells and few plasma cells. However, this compartment had reduced percentages of CD8 alpha+ lymphocytes (28%) and increased percentages of CD4+ lymphocytes (27%) relative to the IEC. However, the LPL CD4:CD8 ratio (1.0) remained low compared with the ratio in peripheral blood. In feline MALT, MHC class II expression was lower than in other peripheral lymphoid compartments. The results of this study provide important reference values for future investigations involving feline intestinal lymphocytes and demonstrates that the leukocyte distribution and phenotypic characteristics of the feline diffuse MALT appear largely similar to the murine, rat and human counterparts.

  7. Functional characterization of tissue-specific enhancers in the DLX5/6 locus. (United States)

    Birnbaum, Ramon Y; Everman, David B; Murphy, Karl K; Gurrieri, Fiorella; Schwartz, Charles E; Ahituv, Nadav


    Disruption of distaless homeobox 5 and 6 (Dlx5/6) in mice results in brain, craniofacial, genital, ear and limb defects. In humans, chromosomal aberrations in the DLX5/6 region, some of which do not encompass DLX5/6, are associated with split hand/foot malformation 1 (SHFM1) as well as intellectual disability, craniofacial anomalies and hearing loss, suggesting that the disruption of DLX5/6 regulatory elements could lead to these abnormalities. Here, we characterized enhancers in the DLX5/6 locus whose tissue-specific expression and genomic location along with previously characterized enhancers correlate with phenotypes observed in individuals with chromosomal abnormalities. By analyzing chromosomal aberrations at 7q21, we refined the minimal SHFM1 critical region and used comparative genomics to select 26 evolutionary conserved non-coding sequences in this critical region for zebrafish enhancer assays. Eight of these sequences were shown to function as brain, olfactory bulb, branchial arch, otic vesicle and fin enhancers, recapitulating dlx5a/6a expression. Using a mouse enhancer assay, several of these zebrafish enhancers showed comparable expression patterns in the branchial arch, otic vesicle, forebrain and/or limb at embryonic day 11.5. Examination of the coordinates of various chromosomal rearrangements in conjunction with the genomic location of these tissue-specific enhancers showed a correlation with the observed clinical abnormalities. Our findings suggest that chromosomal abnormalities that disrupt the function of these tissue-specific enhancers could be the cause of SHFM1 and its associated phenotypes. In addition, they highlight specific enhancers in which mutations could lead to non-syndromic hearing loss, craniofacial defects or limb malformations.

  8. Influence of culture conditions and extracellular matrix alignment on human mesenchymal stem cells invasion into decellularized engineered tissues. (United States)

    Weidenhamer, Nathan K; Moore, Dusty L; Lobo, Fluvio L; Klair, Nathaniel T; Tranquillo, Robert T


    The variables that influence the in vitro recellularization potential of decellularized engineered tissues, such as cell culture conditions and scaffold alignment, have yet to be explored. The goal of this work was to explore the influence of insulin and ascorbic acid and extracellular matrix (ECM) alignment on the recellularization of decellularized engineered tissue by human mesenchymal stem cells (hMSCs). Aligned and non-aligned tissues were created by specifying the geometry and associated mechanical constraints to fibroblast-mediated fibrin gel contraction and remodelling using circular and C-shaped moulds. Decellularized tissues (matrices) of the same alignment were created by decellularization with detergents. Ascorbic acid promoted the invasion of hMSCs into the matrices due to a stimulated increase in motility and proliferation. Invasion correlated with hyaluronic acid secretion, α-smooth muscle actin expression and decreased matrix thickness. Furthermore, hMSCs invasion into aligned and non-aligned matrices was not different, although there was a difference in cell orientation. Finally, we show that hMSCs on the matrix surface appear to differentiate toward a smooth muscle cell or myofibroblast phenotype with ascorbic acid treatment. These results inform the strategy of recellularizing decellularized engineered tissue with hMSCs.

  9. Acoustic radiation force impulse (ARFI) imaging: Characterizing the mechanical properties of tissues using their transient response to localized force (United States)

    Nightingale, Kathryn R.; Palmeri, Mark L.; Congdon, Amy N.; Frinkely, Kristin D.; Trahey, Gregg E.


    Acoustic radiation force impulse (ARFI) imaging utilizes brief, high energy, focused acoustic pulses to generate radiation force in tissue, and conventional diagnostic ultrasound methods to detect the resulting tissue displacements in order to image the relative mechanical properties of tissue. The magnitude and spatial extent of the applied force is dependent upon the transmit beam parameters and the tissue attenuation. Forcing volumes are on the order of 5 mm3, pulse durations are less than 1 ms, and tissue displacements are typically several microns. Images of tissue displacement reflect local tissue stiffness, with softer tissues (e.g., fat) displacing farther than stiffer tissues (e.g., muscle). Parametric images of maximum displacement, time to peak displacement, and recovery time provide information about tissue material properties and structure. In both in vivo and ex vivo data, structures shown in matched B-mode images are in good agreement with those shown in ARFI images, with comparable resolution. Potential clinical applications under investigation include soft tissue lesion characterization, assessment of focal atherosclerosis, and imaging of thermal lesion formation during tissue ablation procedures. Results from ongoing studies will be presented. [Work supported by NIH Grant R01 EB002132-03, and the Whitaker Foundation. System support from Siemens Medical Solutions USA, Inc.

  10. Fabrication and Characterization of Thermoresponsive Polystyrene Nanofibrous Mats for Cultured Cell Recovery (United States)

    Oh, Hwan Hee; Uyama, Hiroshi; Park, Won Ho; Cho, Donghwan; Kwon, Oh Hyeong


    Rapid cell growth and rapid recovery of intact cultured cells are an invaluable technique to maintain the biological functions and viability of cells. To achieve this goal, thermoresponsive polystyrene (PS) nanofibrous mat was fabricated by electrospinning of PS solution, followed by the graft polymerization of thermoresponsive poly(N-isopropylacrylamide)(PIPAAm) on PS nanofibrous mats. Image analysis of the PS nanofiber revealed a unimodal distribution pattern with 400 nm average fiber diameter. Graft polymerization of PIPAAm on PS nanofibrous mats was confirmed by spectroscopic methods such as ATR-FTIR, ESCA, and AFM. Human fibroblasts were cultured on four different surfaces, PIPAAm-grafted and ungrafted PS dishes and PIPAAm-grafted and ungrafted PS nanofibrous mats, respectively. Cells on PIPAAm-grafted PS nanofibrous mats were well attached, spread, and proliferated significantly much more than those on other surfaces. Cultured cells were easily detached from the PIPAAm-grafted surfaces by decreasing culture temperature to 20°C, while negligible cells were detached from ungrafted surfaces. Moreover, cells on PIPAAm-grafted PS nanofibrous mats were detached more rapidly than those on PIPAAm-grafted PS dishes. These results suggest that thermoresponsive nanofibrous mats are attractive cell culture substrates which enable rapid cell growth and recovery from the culture surface for application to tissue engineering and regenerative medicine. PMID:24696851

  11. Diffuse Optical Characterization of the Healthy Human Thyroid Tissue and Two Pathological Case Studies.

    Directory of Open Access Journals (Sweden)

    Claus Lindner

    Full Text Available The in vivo optical and hemodynamic properties of the healthy (n = 22 and pathological (n = 2 human thyroid tissue were measured non-invasively using a custom time-resolved spectroscopy (TRS and diffuse correlation spectroscopy (DCS system. Medical ultrasound was used to guide the placement of the hand-held hybrid optical probe. TRS measured the absorption and reduced scattering coefficients (μa, μs' at three wavelengths (690, 785 and 830 nm to derive total hemoglobin concentration (THC and oxygen saturation (StO2. DCS measured the microvascular blood flow index (BFI. Their dependencies on physiological and clinical parameters and positions along the thyroid were investigated and compared to the surrounding sternocleidomastoid muscle. The THC in the thyroid ranged from 131.9 μM to 144.8 μM, showing a 25-44% increase compared to the surrounding sternocleidomastoid muscle tissue. The blood flow was significantly higher in the thyroid (BFIthyroid = 16.0 × 10-9 cm2/s compared to the muscle (BFImuscle = 7.8 × 10-9 cm2/s, while StO2 showed a small (StO2, muscle = 63.8% to StO2, thyroid = 68.4%, yet significant difference. Two case studies with thyroid nodules underwent the same measurement protocol prior to thyroidectomy. Their THC and BFI reached values around 226.5 μM and 62.8 × 10-9 cm2/s respectively showing a clear contrast to the nodule-free thyroid tissue as well as the general population. The initial characterization of the healthy and pathologic human thyroid tissue lays the ground work for the future investigation on the use of diffuse optics in thyroid cancer screening.

  12. Characterization of TEMPO-oxidized bacterial cellulose scaffolds for tissue engineering applications

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    Luo, Honglin [School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Xiong, Guangyao [School of Mechanical and Electrical Engineering, East China Jiaotong University, Nanchang, Jiangxi 330013 (China); Hu, Da [School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Ren, Kaijing [Department of Joint Surgery, Tianjin Hospital, Tianjin 300211 (China); Yao, Fanglian; Zhu, Yong [School of Chemical Engineering, Tianjin University, Tianjin 300072 (China); Gao, Chuan [School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Wan, Yizao, E-mail: [School of Materials Science and Engineering, Tianjin University, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China)


    Introduction of active groups on the surface of bacterial cellulose (BC) nanofibers is one of the promising routes of tailoring the performance of BC scaffolds for tissue engineering. This paper reported the introduction of aldehyde groups to BC nanofibers by 2,2,6,6-tetramethylpyperidine-1-oxy radical (TEMPO)-mediated oxidation and evaluation of the potential of the TEMPO-oxidized BC as tissue engineering scaffolds. Periodate oxidation was also conducted for comparison. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analyses were carried out to determine the existence of aldehyde groups on BC nanofibers and the crystallinity. In addition, properties relevant to scaffold applications such as morphology, fiber diameter, mechanical properties, and in vitro degradation were characterized. The results indicated that periodate oxidation could introduce free aldehyde to BC nanofibers and the free aldehyde groups on the TEMPO-oxidized BC tended to transfer to acetal groups. It was also found that the advantageous 3D structure of BC scaffolds remained unchanged and that no significant changes in morphology, fiber diameter, tensile structure and in vitro degradation were found after TEMPO-mediated oxidation while significant differences were observed upon periodate oxidation. The present study revealed that TEMPO-oxidation could impart BC scaffolds with new functions while did not degrade their intrinsic advantages. - Highlights: • TEMPO-mediated oxidation on BC scaffold for tissue engineering use was conducted. • TEMPO-mediated oxidation did not degrade the intrinsic advantages of BC scaffold. • TEMPO-mediated oxidation could impart BC scaffold with new functional groups. • Feasibility of TEMPO-oxidized BC as tissue engineering scaffold was confirmed.

  13. Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment.

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    Shirly Sieh

    Full Text Available Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to

  14. PCL/alginate composite scaffolds for hard tissue engineering: fabrication, characterization, and cellular activities. (United States)

    Kim, Yong Bok; Kim, Geun Hyung


    Alginates have been used widely in biomedical applications because of good biocompatibility, low cost, and rapid gelation in the presence of calcium ions. However, poor mechanical properties and fabrication-ability for three-dimensional shapes have been obstacles in hard-tissue engineering applications. To overcome these shortcomings of alginates, we suggest a new composite system, consisting of a synthetic polymer, poly(ε-caprolactone), and various weight fractions (10-40 wt %) of alginate. The fabricated composite scaffolds displayed a multilayered 3D structure, consisting of microsized composite struts, and they provided a 100% offset for each layer. To show the feasibility of the scaffold for hard tissue regeneration, the composite scaffolds fabricated were assessed not only for physical properties, including surface roughness, tensile strength, and water absorption and wetting, but also in vitro osteoblastic cellular responses (cell-seeding efficiency, cell viability, fluorescence analyses, alkaline phosphatase (ALP) activity, and mineralization) by culturing with preosteoblasts (MC3T3-E1). Due to the alginate components in the composites, the scaffolds showed significantly enhanced wetting behavior, water-absorption (∼12-fold), and meaningful biological activities (∼2.1-fold for cell-seeding efficiency, ∼2.5-fold for cell-viability at 7 days, ∼3.4-fold for calcium deposition), compared with a pure PCL scaffold.

  15. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

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    Miessen Katrin


    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.