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Sample records for insect tissue culture

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

    PubMed

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

    2013-01-01

    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. PMID:23940603

  2. Metabolomics Reveals the Heterogeneous Secretome of Two Entomopathogenic Fungi to Ex Vivo Cultured Insect Tissues

    PubMed Central

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

    2013-01-01

    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. PMID:23940603

  3. Insect cell culture and applications to research and pest management

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Building on earlier research, insect cell culture began with the successful establishment of one cell line from pupal ovarian tissue. The field has grown to the extent that now over 500 insect cell lines have been established from many insect species representing numerous insect Orders and from seve...

  4. Recombinant protein production and insect cell culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

    1993-01-01

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

  5. Recombinant Protein Production and Insect Cell Culture and Process

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  6. Tissue Culture in Microgravity

    NASA Technical Reports Server (NTRS)

    Pellis, Neal R.; Duray, Paul H.; Hatfill, Steven J.

    1997-01-01

    Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

  7. Gastric tissue biopsy and culture

    MedlinePlus

    Culture - gastric tissue; Biopsy - gastric tissue ... of organisms that cause infection. A gastric tissue culture may be considered normal if it does not show certain bacteria. Stomach acids normally prevent too much bacteria from growing.

  8. Plant Tissue Culture Studies.

    ERIC Educational Resources Information Center

    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…

  9. NASA Bioreactor tissue culture

    NASA Technical Reports Server (NTRS)

    1998-01-01

    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.

  10. Citrus Tissue Culture

    PubMed Central

    Einset, John W.

    1978-01-01

    In vitro growth of explant (juice vesicle or albedo tissues) cultures from citron (Citrus medica), lemon (C. limon), grapefruit (C. paradisi), sweet orange (C. sinensis), and mandarin (C. reticulata) fruits was stimulated by addition of orange juice (10% v/v optimum) to a basal medium containing Murashige and Skoog salts, 50 grams per liter sucrose, 100 milligrams per liter myo-inositol, 5 milligrams per liter thiamine·HCl, 2 milligrams per liter 2,4-dichlorophenoxyacetic acid and 0.5 milligrams per liter kinetin. In analyzing this effect of orange juice on citron explant cultures, we failed to obtain increased yields by addition of appropriate concentrations of citric acid to the basal medium but obtained growth stimulation when the medium was supplemented with juice from an “acidless” orange variety (cv. Lima). These facts suggest that some component(s) other than citric acid is involved. Addition of the inorganic ash corresponding to 10% (v/v) orange juice to the basal medium had no effect on yields. Similarly, the stimulatory effect of orange juice could not be explained based on its content of sucrose or of organic growth factors already present in the basal medium. ImagesFig. 2 PMID:16660631

  11. Gastric tissue biopsy and culture

    MedlinePlus

    ... laboratory test that examines the tissue sample for bacteria and other organisms that can cause disease. ... of organisms that cause infection. A gastric tissue culture may be ... Stomach acids normally prevent too much bacteria from growing.

  12. Calcium signaling mediates cold sensing in insect tissues

    PubMed Central

    Teets, Nicholas M.; Yi, Shu-Xia; Lee, Richard E.; Denlinger, David L.

    2013-01-01

    The ability to rapidly respond to changes in temperature is a critical adaptation for insects and other ectotherms living in thermally variable environments. In a process called rapid cold hardening (RCH), insects significantly enhance cold tolerance following brief (i.e., minutes to hours) exposure to nonlethal chilling. Although the ecological relevance of RCH is well-established, the underlying physiological mechanisms that trigger RCH are poorly understood. RCH can be elicited in isolated tissues ex vivo, suggesting cold-sensing and downstream hardening pathways are governed by brain-independent signaling mechanisms. We previously provided preliminary evidence that calcium is involved in RCH, and here we firmly establish that calcium signaling mediates cold sensing in insect tissues. In tracheal cells of the freeze-tolerant goldenrod gall fly, Eurosta solidaginis, chilling to 0 °C evoked a 40% increase in intracellular calcium concentration as determined by live-cell confocal imaging. Downstream of calcium entry, RCH conditions significantly increased the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) while reducing phosphorylation of the inhibitory Thr306 residue. Pharmacological inhibitors of calcium entry, calmodulin activation, and CaMKII activity all prevented ex vivo RCH in midgut and salivary gland tissues, indicating that calcium signaling is required for RCH to occur. Similar results were obtained for a freeze-intolerant species, adults of the flesh fly, Sarcophaga bullata, suggesting that calcium-mediated cold sensing is a general feature of insects. Our results imply that insect tissues use calcium signaling to instantly detect decreases in temperature and trigger downstream cold-hardening mechanisms. PMID:23671084

  13. Biotransformations with plant tissue cultures.

    PubMed

    Carew, D P; Bainbridge, T

    1976-01-01

    Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium maculatum were examined for their capacity to transform aniline, anisole, acetanilide, benzoic acid and coumarin. None of the cultures transformed acetanilide but each produced acetanilide when fed aniline. All three cultures converted benzoic acid to its para-hydroxy derivative. Coumarin was selectively hydroxylated at the 7-position by Catharanthus and Conium and anisole was O-demethylated only by older Catharanthus tissue. PMID:1084950

  14. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  15. Application of Tissue Culture in Ornamental Breeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant tissue culture can be broadly defined as the culture of plant cells, tissues, or organs under sterile or aseptic conditions. To most growers, micropropagation is the term that perhaps best describes plant tissue culture. However, plant tissue culture plays an important role through its many ap...

  16. Radiosensitivity of cultured insect cells: II. Diptera

    SciTech Connect

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

  17. Long-term and room temperature operable bioactuator powered by insect dorsal vessel tissue.

    PubMed

    Akiyama, Yoshitake; Iwabuchi, Kikuo; Furukawa, Yuji; Morishima, Keisuke

    2009-01-01

    We present a bioactuator powered by insect dorsal vessel tissue which can work for a long time at room temperature without maintenance. Previously reported bioactuators which exploit contracting ability of mammalian heart muscle cell have required precise environmental control to keep the cell alive and contracting. To overcome this problem, we propose a bioactuator using dorsal vessel tissue. The insect tissue which can grow at room temperature is generally robust over a range of culture conditions compared to mammalian tissues and cells. First, we confirm that a dorsal vessel tissue of lepidoptera larva Ctenoplusia agnata contracts spontaneously for at least 30 days without medium replacement at 25 degrees C. Using the dorsal vessel tissue cultured under the same conditions, we succeed in driving micropillars 100 microm in diameter and 1000 microm in height for more than 90 days. The strongest displacement of the micropillar top occurs on the 42(nd) day and is 23 microm. Based on these results, the contracting force is roughly estimated as 4.7 microN which is larger than that by a few mammalian cardiomyocytes (3.4 microN). Definite displacements of more than 10 microm are observed for 58 days from the 15(th) to the 72(nd) days. The number of life cycles can be roughly calculated as 7.5 x 10(5) times for the average frequency of about 0.15 Hz, which is no less than that of conventional mechanical actuators. These results suggest that the insect dorsal vessel tissue is a more promising material for bioactuators used at room temperature than other biological cell-based materials. PMID:19209346

  18. Three Dimensional Optic Tissue Culture and Process

    NASA Technical Reports Server (NTRS)

    OConnor, Kim C. (Inventor); Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); Aten, Laurie A. (Inventor); Francis, Karen M. (Inventor); Caldwell, Delmar R. (Inventor); Prewett, Tacey L. (Inventor); Fitzgerald, Wendy S. (Inventor)

    1999-01-01

    A process for artificially producing three-dimensional optic tissue has been developed. The optic cells are cultured in a bioireactor at low shear conditions. The tissue forms as normal, functional tissue grows with tissue organization and extracellular matrix formation.

  19. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture.

    PubMed

    Shirk, Paul D; Furlong, Richard B

    2016-01-01

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity. PMID:26794473

  20. Three dimensional optic tissue culture and process

    NASA Technical Reports Server (NTRS)

    Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor); Goodwin, Thomas J. (Inventor); Francis, Karen M. (Inventor); Cardwell, Delmar R. (Inventor); Oconnor, Kim (Inventor); Fitzgerald, Wendy S. (Inventor); Aten, Laurie A. (Inventor)

    1994-01-01

    A process for artificially producing three-dimensional optic tissue has been developed. The optic cells are cultured in a bioreactor at low shear conditions. The tissue forms normal, functional tissue organization and extracellular matrix.

  1. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

    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 cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  2. Optical metabolic imaging of live tissue cultures

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Cook, Rebecca S.; Arteaga, Carlos L.; Skala, Melissa C.

    2013-02-01

    The fluorescence properties, both intensity and fluorescence lifetime, of NADH and FAD, two coenzymes of metabolism, are sensitive, high resolution measures of cellular metabolism. However, often in vivo measurements of tissue are not feasible. In this study, we investigate the stability over time of two-photon auto-fluorescence imaging of NADH and FAD in live-cultured tissues. Our results demonstrate that cultured tissues remain viable for at least several days post excision. Furthermore, the optical redox ratio, NADH fluorescence lifetime, and FAD fluorescence lifetime do not significantly change in the cultured tissues over time. With these findings, we demonstrate the potential of sustained tissue culture techniques for optical metabolic imaging.

  3. Plant Tissue Culture in a Bag.

    ERIC Educational Resources Information Center

    Beck, Mike

    2000-01-01

    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)

  4. Tissue culture media and reagents*

    PubMed Central

    Perkins, F. T.

    1973-01-01

    The various factors that affect the growth of cell cultures in vitro are considered and suggestions are made as to where the standardization of components would help to provide more uniform products. The manufacture of powdered media in bulk and the establishment of cell banks have been major advances, but much research is needed for the standardization or replacement of serum. PMID:4206452

  5. Chlamydial Infection of Conjunctival Tissues in Culture

    PubMed Central

    Moore, J. E.; Griffiths, M. S.; Pearce, J. H.

    1974-01-01

    Baboon conjunctival cells removed as scrapings from the conjunctiva could be disaggregated and maintained in culture for up to 5 days without appreciable loss of viability. Attempts to infect cultures with a TRIC agent or its “fast” variant were unsuccessful. Nevertheless such cultures appeared to support the growth of TRIC agent since conjunctival cells obtained from infected baboons developed inclusions while maintained in vitro. Cell viability and tissue structure were preserved over 6 days organ culture of conjunctivae. Organ cultures supported the growth of chlamydiae: the TRIC fast variant grew in low titre in baboon cultures; the highly virulent gp-ic agent grew to high titre in guinea-pig cultures. Frequent inclusion bodies and damaged epithelium were seen in histological examination of infected guinea-pig cultures; occasional sub-epithelial inclusions were detected, some of which were atypical in morphology. ImagesFigs. 1-6 PMID:4139967

  6. Activation of tobacco retrotransposons during tissue culture.

    PubMed Central

    Hirochika, H

    1993-01-01

    Sequences of at least three new families of retrotransposons (Tto1-Tto3) were amplified by PCR from cDNA prepared from protoplasts of an established tobacco cell line, based on the fact that certain amino acids are highly conserved in the reverse transcriptases encoded by retrotransposons. Structural analysis indicates that Tto1 is 5.5 kb long and has features typical of retrotransposons. Transcription of Tto1 starting in the long terminal repeat was active only in cultured cells. Protoplast formation enhanced the transcription. The copy number of Tto1 increased 10-fold in established cell lines; it also increased in plants regenerated from tissue cultures and in transgenic plants. These results indicate that Tto1 is activated during tissue culture. This is the first demonstration of activation of a plant retrotransposon by tissue culture. The copy number of Tto2 and a previously isolated transposon, Tnt1, also increased in established cell lines, indicating that these two retrotransposons may also be activated by tissue culture. These three retrotransposons are cryptic in normally propagated plants: no difference in the copy number was observed between individuals of the same cultivars or even between different cultivars. Images PMID:8389699

  7. Proteoglycan modifications by granulation tissue in culture.

    PubMed

    Quintner, M I; Kollar, E J; Rossomando, E F

    1982-01-01

    To study the process of tissue remodeling that occurs during wound healing, radioactive proteoglycan ([35S]-PGS) was used to assay for enzymatic activities present in the extracellular fluid of healing tissue. Mice, wounded by removal of a 2 x 1.5 cm patch of skin from the dorsal surface, were sacrificed after 3 days of healing. Granulation tissue (1 cm2) was removed, spread onto a sterile wire mesh support and placed in the center well of an organ culture dish. To each well was added 1 ml MCDB medium supplemented with 10% fetal calf serum and antibiotics and 5-20 microliters of [35S]-PGS (100,000 cpm/10 microliters). Medium, removed from the well by aspiration after 24 and 48 h of culture, was boiled 5 min at 100 degrees C and stored frozen at -20 degrees C. Alterations of the PGS were assayed with a Sepharose 4B column (1 x 50 cm) which had an excluded and included volume of 17 and 46 ml, respectively. PGS, incubated without cells or with tissues from unwounded animals, eluted at 26 ml. PGS, incubated with granulation tissue and cultured for either 24 or 48 h, eluted from the Sepharose 4B at 29 ml, a 10% increase in elution volume, suggesting that the size or shape of the PGS has been altered by enzymes secreted by the cells of the granulation tissue. In contrast, PGS incubated with tissues from unwounded animals or without granulation tissue showed no changes. These data suggest that enzymatic activities secreted by cells of granulation tissue may be involved in remodeling during healing. PMID:6749574

  8. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  9. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 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 culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  10. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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 culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  11. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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 culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  12. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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 culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  13. Murine trabecular meshwork cells in tissue culture.

    PubMed

    Begley, C G; Yue, B Y; Hendricks, R L

    1991-11-01

    Trabecular meshwork cells from an inbred strain of mice (A/J) were established in tissue culture. Within 1 hour of enucleation, tissue containing the cornea and the chamber angle was excised and placed in tissue culture. Two to five days later, three cell types grew from the explants. Two of these cell types, corneal endothelium and fibroblasts, grew together, with the fibroblasts preferentially spreading on top of the endothelial cells. The trabecular meshwork cells extended from the explant as a distinct morphological type. The corneal endothelium and its associated fibroblasts were then removed from the culture flask with a sterile cotton swab, leaving a monolayer of pure trabecular meshwork cells. These cells required 3-4 weeks to reach confluency and could be passaged five times. They were actively phagocytic in culture and exhibited immunoreactivity to antibodies against two extracellular matrix components, laminin and collagen type IV. Mouse trabecular meshwork cells also expressed receptors for acetylated low-density lipoprotein, a property shared by trabecular meshwork cells derived from other species. The availability of trabecular meshwork cells from an inbred strain of mice will facilitate future in vivo functional studies of these cells in a syngeneic system, as well as investigations of potential immunoregulatory properties of the trabecular meshwork. PMID:1782800

  14. Tissue engineering of cultured skin substitutes.

    PubMed

    Horch, Raymund E; Kopp, Jürgen; Kneser, Ulrich; Beier, Justus; Bach, Alexander D

    2005-01-01

    Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin. PMID:16202208

  15. Pathogen Propagation in Cultured Three-Dimensional Tissue Mass

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.

  16. Pathogen propagation in cultured three-dimensional tissue mass

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

    A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.

  17. Transferring isolated mitochondria into tissue culture cells

    PubMed Central

    Yang, Yi-Wei; Koob, Michael D.

    2012-01-01

    We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained. PMID:22753025

  18. Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System.

    PubMed

    Kruithof, Boudewijn P T; Lieber, Samuel C; Kruithof-de Julio, Marianna; Gaussin, Vincian; Goumans, Marie José

    2015-01-01

    Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve. PMID:26555276

  19. Ecosystem engineering and manipulation of host plant tissues by the insect borer Oncideres albomarginata chamela.

    PubMed

    Calderón-Cortés, Nancy; Uribe-Mú, Claudia A; Martínez-Méndez, A Karen; Escalera-Vázquez, Luis H; Cristobal-Pérez, E Jacob; García-Oliva, Felipe; Quesada, Mauricio

    2016-01-01

    Ecosystem engineering by insect herbivores occurs as the result of structural modification of plants manipulated by insects. However, only few studies have evaluated the effect of these modifications on the plant responses induced by stem-borers that act as ecosystem engineers. In this study, we evaluated the responses induced by the herbivory of the twig-girdler beetle Oncideres albomarginata chamela (Cerambycidae: Lamiinae) on its host plant Spondias purpurea (Anacardiaceae), and its relationship with the ecosystem engineering process carried out by this stem-borer. Our results demonstrated that O. albomarginata chamela branch removal induced the development of lateral branches increasing the resources needed for the development of future insect generations, of its own offspring and of many other insect species. Detached branches represent habitats with high content of nitrogen and phosphorous, which eventually can be incorporated into the ecosystem, increasing nutrient cycling efficiency. Consequently, branch removal and the subsequent plant tissue regeneration induced by O. albomarginata chamela represent key mechanisms underlying the ecosystem engineering process carried out by this stem-borer, which enhances arthropod diversity in the ecosystem. PMID:26654885

  20. Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore.

    PubMed

    Zagrobelny, Mika; Olsen, Carl Erik; Pentzold, Stefan; Fürstenberg-Hägg, Joel; Jørgensen, Kirsten; Bak, Søren; Møller, Birger Lindberg; Motawia, Mohammed Saddik

    2014-01-01

    Considering the staggering diversity of bioactive natural products present in plants, insects are only able to sequester a small number of phytochemicals from their food plants. The mechanisms of how only some phytochemicals are sequestered and how the sequestration process takes place remains largely unknown. In this study the model system of Zygaena filipendulae (Lepidoptera) and their food plant Lotus corniculatus is used to advance the knowledge of insect sequestration. Z. filipendulae larvae are dependent on sequestration of the cyanogenic glucosides linamarin and lotaustralin from their food plant, and have a much lower fitness if reared on plants without these compounds. This study investigates the fate of the cyanogenic glucosides during ingestion, sequestration in the larvae, and in the course of insect ontogeny. To this purpose, double-labeled linamarin and lotaustralin were chemically synthesized carrying two stable isotopes, a (2)H labeled aglucone and a (13)C labeled glucose moiety. In addition, a small amount of (14)C was incorporated into the glucose residue. The isotope-labeled compounds were applied onto cyanogenic L. corniculatus leaves that were subsequently presented to the Z. filipendulae larvae. Following ingestion by the larvae, the destiny of the isotope labeled cyanogenic glucosides was monitored in different tissues of larvae and adults at selected time points, using radio-TLC and LC-MS analyses. It was shown that sequestered compounds are taken up intact, contrary to earlier hypotheses where it was suggested that the compounds would have to be hydrolyzed before transport across the gut. The uptake from the larval gut was highly stereo selective as the β-glucosides were retained while the α-glucosides were excreted and recovered in the frass. Sequestered compounds were rapidly distributed into all analyzed tissues of the larval body, partly retained throughout metamorphosis and transferred into the adult insect where they were

  1. Cell and tissue culture of Miscanthus Sacchariflorus

    SciTech Connect

    Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K.

    1995-11-01

    Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerants of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.

  2. A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

    PubMed Central

    Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

    2014-01-01

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. PMID:25486605

  3. Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana.

    PubMed

    Lu, Rui-Li; Bao, Guan-Hu; Hu, Feng-Lin; Huang, Bo; Li, Chun-Ru; Li, Zeng-Zhi

    2014-02-15

    A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD₅₀) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 μg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time. PMID:24128585

  4. Development of germ-free plants and tissue culture

    NASA Technical Reports Server (NTRS)

    Venketeswaran, S.

    1973-01-01

    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.

  5. Tissue-Specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus planipennis)

    PubMed Central

    Mittapalli, Omprakash; Bai, Xiaodong; Bonello, Pierluigi; Herms, Daniel A.

    2010-01-01

    Background The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level. Methodology and Principal Findings Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level. Conclusions and Significance To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis. PMID:21060843

  6. Potential for forest tree improvement via tissue culture

    SciTech Connect

    Karnosky, D.F.

    1981-02-01

    The culture of cells, tissues, and organs in vitro offers unparalleled opportunity for forest tree improvement. Vegetative propagation of selected superior genotypes and hybrids, production and culture of haploids, asexual hybridization via protoplast fusion, freeze preservation of valuable genotypes, and the selection of cell lines tolerant to stresses such as diseases, drought, heavy metals, or salts through tissue culture may someday provide forest geneticists efficient and economical methods to supplement tree improvement programs. Heat treatments and meristem culture currently provide a pratical means of eliminating harmful virus and mycoplasma diseases from vegatively propagated trees. For the most part, however, the forest tree tissue culture research is only in its infancy. Research must be expanded to realize the full potential available from tissue culture. Considerable effort will be necessary to solve the many problems now deterring practical use of tissue culture in forest tree improvement and reforestation programs. (Refs. 93).

  7. Study Progress on Tissue Culture of Maize Mature Embryo

    NASA Astrophysics Data System (ADS)

    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.

  8. Electrical stimulation of cultured lepidopteran dorsal vessel tissue: an experiment for development of bioactuators.

    PubMed

    Akiyama, Yoshitake; Iwabuchi, Kikuo; Furukawa, Yuji; Morishima, Keisuke

    2010-05-01

    An insect dorsal vessel (DV) is well suited for a bioactuator since it is capable of contracting autonomously, and its tissue and cells are more environmentally robust under culturing conditions compared with mammalian tissue. In this study, electrical pulse stimulation was examined so as to regulate a bioactuator using the DV tissue. The DV tissue of a larva of Ctenoplusia agnate was assembled on a micropillar array, which was stimulated after culturing for about 3 wk. The contraction of the DV tissue was evaluated by image analysis to measure lateral displacements at the micropillar top. As a result, suitable stimulation conditions in a 35-mm petri dish were determined as: applied voltage of 10 V with 20-ms duration. Next, the time lag between the onset of electrical stimulus and the onset of mechanical contraction (electromechanical delay (EMD)) was estimated. A light-emitting diode (LED) was connected serially with the petri dish, and the LED flashed when electrical pulses were given. Movie images were analyzed in which electrical pulses made the DV tissue contract and the LED flashed virtually simultaneously; from these, the EMD was estimated as approximately 50 ms. These results suggest that the electrical pulse stimulation is capable of regulating the DV tissue, and the micropillar array is a useful biological tool to investigate physiological properties of muscle tissue. PMID:20063072

  9. Effect of lunar materials on plant tissue culture.

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

    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.

  10. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-11-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18428384

  11. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18265370

  12. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  13. Techniques for mammalian cell tissue culture.

    PubMed

    Phelan, Mary C

    2006-12-01

    This appendix opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18429293

  14. An insect TEP in a crustacean is specific for cuticular tissues and involved in intestinal defense.

    PubMed

    Wu, Chenglin; Noonin, Chadanat; Jiravanichpaisal, Pikul; Söderhäll, Irene; Söderhäll, Kenneth

    2012-02-01

    In an attempt to identify genes encoding thioester-containing proteins in the freshwater crayfish, Pacifastacus leniusculus, three different cDNAs were found. A phylogenetic analysis of these proteins indicates that they can be classified into two subfamilies: two alpha-2-macroglobulins (Pl-A2M1, Pl-A2M2) showing a close similarity to shrimp A2M, and one insect TEP-like protein (Pl-TEP). This is the first report of an insect TEP-like protein in a crustacean. Crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP cDNAs encode proteins with 1480, 1586 or 1507 amino acids, respectively. Pl-A2M1, Pl-A2M2 and Pl-TEP have the basic domain structure and functionally important residues for each molecule, and their mRNA was detected in different parts of the body, suggesting that they may have different functions. Pl-A2M1 was mainly expressed in hemocytes and Pl-A2M2 was highly expressed in heart and nerve, while Pl-TEP was exclusively expressed in cuticular tissues such as gill and intestine. RNA interference of Pl-TEP in vivo resulted in that these animals were slightly less resistant when fed with the bacterium, Pseudomonas libanensis/gessardii. Furthermore, when TEP activity was blocked using methylamine followed by bacterial feeding, the animals were killed to a higher extent compared to a control group. Taken together, this indicates that Pl-TEP and/or Pl-A2M1, Pl-A2M2 may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues. PMID:22193393

  15. Explant culture of sarcoma patients' tissue.

    PubMed

    Muff, Roman; Botter, Sander M; Husmann, Knut; Tchinda, Joelle; Selvam, Philomina; Seeli-Maduz, Franziska; Fuchs, Bruno

    2016-07-01

    Human sarcomas comprise a heterogeneous group of rare tumors that affect soft tissues and bone. Due to the scarcity and heterogeneity of these diseases, patient-derived cells that can be used for preclinical research are limited. In this study, we investigated whether the tissue explant technique can be used to obtain sarcoma cell lines from fresh as well as viable frozen tissue obtained from 8 out of 12 soft tissue and 9 out of 13 bone tumor entities as defined by the World Health Organization. The success rate, defined as the percent of samples that yielded sufficient numbers of outgrowing cells to be frozen, and the time to freeze were determined for a total of 734 sarcoma tissue specimens. In 552 cases (75%) enough cells were obtained to be frozen at early passage. Success rates were higher in bone tumors (82%) compared with soft tissue tumors (68%), and the mean time to freezing was lower in bone tumors (65 days) compared with soft tissue tumors (84 days). Overall, from 40% of the tissues cells could be frozen at early passage within <2 month after tissue removal. Comparable results as with fresh tissue were obtained after explant of viable frozen patient-derived material. In a selected number of bone and soft tissue sarcoma entities, conventional karyotyping and/or FISH (fluorescence in situ hybridization) analysis revealed a high amount (>60%) of abnormal cells in 41% of analyzed samples, especially in bone sarcomas (osteosarcoma and Ewing sarcoma). In conclusion, the explant technique is well suited to establish patient-derived cell lines for a large majority of bone and soft tissue sarcoma entities with adequate speed. This procedure thus opens the possibility for molecular analysis and drug testing for therapeutic decision making even during patient treatment. PMID:27111283

  16. Plant Tissue Cultures of Juniperus virginiana.

    PubMed

    Kašparová, Marie; Spilková, Jirina; Cvak, Ladislav; Siatka, Tomáš; Martin, Jan

    2016-05-01

    Callus cultures of Juniperus virginiana L. (varieties 'Hetzii', 'Glauca', 'Grey Owl') were derived from fresh leaves of garden-grown trees on Schenk and Hildebrandt medium supplemented with 3.0 mg/L of α-naphthaleneacetic acid, 0.2 mg/L of kinetin and 15 mg/L of ascorbic acid. The growth characteristics of one-year-old and two-years-old cultures were determined. The maximum biomass in all varieties was achieved on the 35th day of the cultivation period. The increase in fresh weights of two-years-old callus cultures, when compared with one-year-old callus cultures, was as follows: variety 'Hetzii' by 25%, variety 'Glauca' by 29% and variety 'Grey Owl' by 49%. J. virginiana suspension cultures (varieties 'Hetzii', 'Glauca', 'Grey Owl') were derived from two-years-old callus cultures on Schenk and Hildebrandt medium supplemented with 3.0 mg/L of α-naphthaleneacetic acid, 0.2 mg/L of kinetin and 15 mg/L of ascorbic acid. The maximum biomass of all varieties was found on the 21st day of the cultivation period. These results indicate that a sub-cultivation interval of 35 days for callus cultures and of 21st days for suspension cultures can be recommended. The callus and suspension cultures of J. virginiana of the variety 'Glauca' have the best survivability and thus provide the most biomass. PMID:27319150

  17. Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein

    PubMed Central

    Jia, Dongsheng; Chen, Hongyan; Zheng, Ailing; Chen, Qian; Liu, Qifei; Xie, Lianhui

    2012-01-01

    An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins. PMID:22398296

  18. Cytological studies of lunar treated tissue cultures

    NASA Technical Reports Server (NTRS)

    Halliwell, R. S.

    1972-01-01

    An electron microscopic study was made of botanical materials, particularly pine tissues, treated with lunar materials collected by Apollo 12 quarantine mission. Results show unusual structural changes within several of the treated tissues. The bodies, as yet unidentified, resemble virus particles observed within infected plant cells. Although the size and shape of the structures are comparable to rod shaped virus particles such as Tobacco mosaic, the numerical distribution, affinity for stains, and intercellular location are different.

  19. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    ERIC Educational Resources Information Center

    Bozzone, Donna M.

    1997-01-01

    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)

  20. Three-dimensional tissue culture based on magnetic cell levitation

    NASA Astrophysics Data System (ADS)

    Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.; Killian, T. C.; Arap, Wadih; Pasqualini, Renata

    2010-04-01

    Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

  1. Explant exenisation for tissue culture in marine macroalgae

    NASA Astrophysics Data System (ADS)

    Liu, Xuewu; Kloareg, Bernard

    1992-09-01

    Unialgal explants from Laminaria digitata, and from a variety of red algae, were obtained by hand removing the visible epiphytes, and stirring the tissue in the presence of glass beads. Two antibiotic mixtures were found to be efficient in removing the contaminating fungi and bacteria from the algae. The procedure proved suitable as a primary step in the tissue culture of the investigated species.

  2. A heated stage and tissue culture chamber for electrophysiology.

    PubMed

    Bonkowski, L; Runion, H I

    1976-12-15

    A simple, inexpensive heating circuit is described for use with a warmed microscope stage, small tissue bath, and heated tissue culture chamber. A major consideration in the design of the apparatus is a compatability with electrophysiological studies. Thus, proper shielding and low profile for microelectrode positioning are featured. PMID:1021472

  3. Tissue-Culture Method of Cloning Rubber Plants

    NASA Technical Reports Server (NTRS)

    Ball, E. A.

    1983-01-01

    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.

  4. Microfluidic 3D cell culture: from tools to tissue models.

    PubMed

    van Duinen, Vincent; Trietsch, Sebastiaan J; Joore, Jos; Vulto, Paul; Hankemeier, Thomas

    2015-12-01

    The transition from 2D to 3D cell culture techniques is an important step in a trend towards better biomimetic tissue models. Microfluidics allows spatial control over fluids in micrometer-sized channels has become a valuable tool to further increase the physiological relevance of 3D cell culture by enabling spatially controlled co-cultures, perfusion flow and spatial control over of signaling gradients. This paper reviews most important developments in microfluidic 3D culture since 2012. Most efforts were exerted in the field of vasculature, both as a tissue on its own and as part of cancer models. We observe that the focus is shifting from tool building to implementation of specific tissue models. The next big challenge for the field is the full validation of these models and subsequently the implementation of these models in drug development pipelines of the pharmaceutical industry and ultimately in personalized medicine applications. PMID:26094109

  5. Tissue Cultures Derived from Ineffective Root Nodules of Alfalfa 1

    PubMed Central

    Vance, Carroll P.; Johnson, Lois E. B.; Boylan, Kristin L. M.

    1984-01-01

    Callus tissue cultures were developed from apical meristem regions of tumor-like ineffective root nodules of alfalfa. Callus growth was a function of tissue source and hormone composition and concentration. Callus derived from ineffective nodules also were shown not to contain Rhizobium meliloti. Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase and phosphoenolpyruvate carboxylase activities were present in callus cultures and in the respective nodule source used for callus induction. The mean specific activity of all enzymes evaluated was higher in callus cultures than in ineffective nodules. Quantitative but not qualitative differences in enzyme activities were evident between ineffective nodules and callus derived from these nodules. Tissue cultures derived from ineffective nodules may provide a model system to evaluate host plant-Rhizobium interactions. Images Fig. 1 PMID:16663985

  6. Extraction and assembly of tissue-derived gels for cell culture and tissue engineering.

    PubMed

    Uriel, Shiri; Labay, Edwardine; Francis-Sedlak, Megan; Moya, Monica L; Weichselbaum, Ralph R; Ervin, Natalia; Cankova, Zdravka; Brey, Eric M

    2009-09-01

    Interactions with the extracellular matrix (ECM) play an important role in regulating cell function. Cells cultured in, or on, three-dimensional ECM recapitulate similar features to those found in vivo that are not present in traditional two-dimensional culture. In addition, both natural and synthetic materials containing ECM components have shown promise in a number of tissue engineering applications. Current materials available for cell culture and tissue engineering do not adequately reflect the diversity of ECM composition between tissues. In this paper, a method is presented for extracting solutions of proteins and glycoproteins from soft tissues and inducing assembly of these proteins into gels. The extracts contain ECM proteins specific to the tissue source with low levels of intracellular molecules. Gels formed from the tissue-derived extracts have nanostructure similar to ECM in vivo and can be used to culture cells as both a thin substrate coating and a thick gel. This technique could be used to assemble hydrogels with varying composition depending upon the tissue source, hydrogels for three-dimensional culture, as scaffolds for tissue engineering therapies, and to study cell-matrix interactions. PMID:19115821

  7. Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

    PubMed Central

    Uriel, Shiri; Labay, Edwardine; Francis-Sedlak, Megan; Moya, Monica L.; Weichselbaum, Ralph R.; Ervin, Natalia; Cankova, Zdravka

    2009-01-01

    Interactions with the extracellular matrix (ECM) play an important role in regulating cell function. Cells cultured in, or on, three-dimensional ECM recapitulate similar features to those found in vivo that are not present in traditional two-dimensional culture. In addition, both natural and synthetic materials containing ECM components have shown promise in a number of tissue engineering applications. Current materials available for cell culture and tissue engineering do not adequately reflect the diversity of ECM composition between tissues. In this paper, a method is presented for extracting solutions of proteins and glycoproteins from soft tissues and inducing assembly of these proteins into gels. The extracts contain ECM proteins specific to the tissue source with low levels of intracellular molecules. Gels formed from the tissue-derived extracts have nanostructure similar to ECM in vivo and can be used to culture cells as both a thin substrate coating and a thick gel. This technique could be used to assemble hydrogels with varying composition depending upon the tissue source, hydrogels for three-dimensional culture, as scaffolds for tissue engineering therapies, and to study cell–matrix interactions. PMID:19115821

  8. Influence of simulated microgravity on the longevity of insect-cell culture

    NASA Technical Reports Server (NTRS)

    Cowger, N. L.; O'Connor, K. C.; Bivins, J. E.

    1997-01-01

    Simulated microgravity within the NASA High Aspect Rotating-Wall Vessel (HARV) provides a quiescent environment to culture fragile insect cells. In this vessel, the duration of stationary and death phase for cultures of Spodoptera frugiperda cells was greatly extended over that achieved in shaker-flask controls. For both HARV and control cultures, S. frugiperda cells grew to concentrations in excess of 1 x 10(7) viable cells ml-1 with viabilities greater than 90%. In the HARV, stationary phase was maintained 9-15 days in contrast to 4-5 days in the shaker flask. Furthermore, the rate of cell death was reduced in the HARV by a factor of 20-90 relative to the control culture and was characterized with a death rate constant of 0.01-0.02 day-1. Beginning in the stationary phase and continuing in the death phase, there was a significant decrease in population size in the HARV versus an increase in the shaker flask. This phenomenon could represent cell adaptation to simulated microgravity and/or a change in the ratio of apoptotic to necrotic cells. Differences observed in this research between the HARV and its control were attributed to a reduction in hydrodynamic forces in the microgravity vessel.

  9. Tissue culture apparatus for flight experimentation

    NASA Technical Reports Server (NTRS)

    Scheld, H. W.; Magnuson, J. W.; Krikorian, A. D.

    1985-01-01

    The development of an apparatus for in-flight treatment of cells, tissues, or small organisms for microscopic and chemical analyses is discussed. The hardware for the apparatus is to have: (1) automated functions, (2) the capability to interface with ground-based facilities, (3) independently controlled chambers, (4) variable chamber configurations and volumes, and (4) the capabilities for processing the materials. The components of the equipment used on Skylab 3 for the study of animal cells are described. The design of an apparatus which incorporates all the required capabilities is proposed.

  10. Differential metabolic responses of Beauveria bassiana cultured in pupae extracts, root exudates and its interactions with insect and plant.

    PubMed

    Luo, Feifei; Wang, Qian; Yin, Chunlin; Ge, Yinglu; Hu, Fenglin; Huang, Bo; Zhou, Hong; Bao, Guanhu; Wang, Bin; Lu, Ruili; Li, Zengzhi

    2015-09-01

    Beauveria bassiana is a kind of world-wide entomopathogenic fungus and can also colonize plant rhizosphere. Previous researches showed differential expression of genes when entomopathogenic fungi are cultured in insect or plant materials. However, so far there is no report on metabolic alterations of B. bassiana in the environments of insect or plant. The purpose of this paper is to address this problem. Herein, we first provide the metabolomic analysis of B. bassiana cultured in insect pupae extracts (derived from Euproctis pseudoconspersa and Bombyx mori, EPP and BMP), plant root exudates (derived from asparagus and carrot, ARE and CRE), distilled water and minimal media (MM), respectively. Principal components analysis (PCA) shows that mycelia cultured in pupae extracts and root exudates are evidently separated and individually separated from MM, which indicates that fungus accommodates to insect and plant environments by different metabolic regulation mechanisms. Subsequently, orthogonal projection on latent structure-discriminant analysis (OPLS-DA) identifies differential metabolites in fungus under three environments relative to MM. Hierarchical clustering analysis (HCA) is performed to cluster compounds based on biochemical relationships, showing that sphingolipids are increased in BMP but are decreased in EPP. This observation further implies that sphingolipid metabolism may be involved in the adaptation of fungus to different hosts. In the meantime, sphingolipids are significantly decreased in root exudates but they are not decreased in distilled water, suggesting that some components of the root exudates can suppress sphingolipid to down-regulate sphingolipid metabolism. Pathway analysis finds that fatty acid metabolism is maintained at high level but non-ribosomal peptides (NRP) synthesis is unaffected in mycelia cultured in pupae extracts. In contrast, fatty acid metabolism is not changed but NRP synthesis is high in mycelia cultured in root exudates

  11. Specimen Sample Preservation for Cell and Tissue Cultures

    NASA Technical Reports Server (NTRS)

    Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

    1996-01-01

    The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

  12. The role of silicon in plant tissue culture.

    PubMed

    Sivanesan, Iyyakkannu; Park, Se Won

    2014-01-01

    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. PMID:25374578

  13. The role of silicon in plant tissue culture

    PubMed Central

    Sivanesan, Iyyakkannu; Park, Se Won

    2014-01-01

    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. PMID:25374578

  14. Micromolded Gelatin Hydrogels for Extended Culture of Engineered Cardiac Tissues

    PubMed Central

    McCain, Megan L.; Agarwal, Ashutosh; Nesmith, Haley W.; Nesmith, Alexander P.; Parker, Kevin Kit

    2014-01-01

    Defining the chronic cardiotoxic effects of drugs during preclinical screening is hindered by the relatively short lifetime of functional cardiac tissues in vitro, which are traditionally cultured on synthetic materials that do not recapitulate the cardiac microenvironment. Because collagen is the primary extracellular matrix protein in the heart, we hypothesized that micromolded gelatin hydrogel substrates tuned to mimic the elastic modulus of the heart would extend the lifetime of engineered cardiac tissues by better matching the native chemical and mechanical microenvironment. To measure tissue stress, we used tape casting, micromolding, and laser engraving to fabricate gelatin hydrogel muscular thin film cantilevers. Neonatal rat cardiac myocytes adhered to gelatin hydrogels and formed aligned tissues as defined by the microgrooves. Cardiac tissues could be cultured for over three weeks without declines in contractile stress. Myocytes on gelatin had higher spare respiratory capacity compared to those on fibronectin-coated PDMS, suggesting that improved metabolic function could be contributing to extended culture lifetime. Lastly, human induced pluripotent stem cell-derived cardiac myocytes adhered to micromolded gelatin surfaces and formed aligned tissues that remained functional for four weeks, highlighting their potential for human-relevant chronic studies. PMID:24731714

  15. Practical Instruction in Tissue Culture and Cytogenetics for Sandwich Students.

    ERIC Educational Resources Information Center

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

    1973-01-01

    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)

  16. Immunomodulatory potential of shatavarins produced from Asparagus racemosus tissue cultures

    PubMed Central

    Pise, Mashitha Vinod; Rudra, Jaishree Amal; Upadhyay, Avinash

    2015-01-01

    Medicinal properties of Asparagus racemosus (vernacular name: Shatavari) are attributed to its steroidal saponins called shatavarins. This plant is facing the threat of being endangered due to several developmental, seasonal constrains and malpractices involved in its collection and storage. To support its conservation, a tissue culture protocol is standardized which produces 20 fold higher levels of shatavarin. Here we evaluate the bioactivity and immunomodulatory potential of in vitro produced shatavarins from cell cultures of AR using human peripheral blood lymphocytes. In vitro produced shatavarin stimulated immune cell proliferation and IgG secretion in a dose dependent manner. It stimulated interleukin (IL)-12 production and inhibited production of IL-6. It also had strong modulatory effects on Th1/Th2 cytokine profile, indicating its potential application for immunotherapies where Th1/Th2 balance is envisaged. Our study demonstrating the bioactivity of tissue cultured AR extracts supports further in vivo evaluation of its immunomodulatory efficacy. PMID:26283842

  17. Immunomodulatory potential of shatavarins produced from Asparagus racemosus tissue cultures.

    PubMed

    Pise, Mashitha Vinod; Rudra, Jaishree Amal; Upadhyay, Avinash

    2015-01-01

    Medicinal properties of Asparagus racemosus (vernacular name: Shatavari) are attributed to its steroidal saponins called shatavarins. This plant is facing the threat of being endangered due to several developmental, seasonal constrains and malpractices involved in its collection and storage. To support its conservation, a tissue culture protocol is standardized which produces 20 fold higher levels of shatavarin. Here we evaluate the bioactivity and immunomodulatory potential of in vitro produced shatavarins from cell cultures of AR using human peripheral blood lymphocytes. In vitro produced shatavarin stimulated immune cell proliferation and IgG secretion in a dose dependent manner. It stimulated interleukin (IL)-12 production and inhibited production of IL-6. It also had strong modulatory effects on Th1/Th2 cytokine profile, indicating its potential application for immunotherapies where Th1/Th2 balance is envisaged. Our study demonstrating the bioactivity of tissue cultured AR extracts supports further in vivo evaluation of its immunomodulatory efficacy. PMID:26283842

  18. The role of activated charcoal in plant tissue culture.

    PubMed

    Thomas, T Dennis

    2008-01-01

    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. PMID:18786626

  19. In vitro ovarian tissue and organ culture: a review.

    PubMed

    Devine, P J; Rajapaksa, K S; Hoyer, Patricia B

    2002-09-01

    Many investigations have utilized techniques for culturing ovarian tissue or isolated follicles in vitro. Whole ovaries from fetal or neonatal rodents have been incubated in organ culture systems. This has been utilized to understand the sequence of follicle formation and its hormonal requirements, activation of quiescent follicles, follicular growth and development, and acquisition of steroidogenic capabilities. Adaptations of this technique include incubation of ovaries in a chamber continuously perfused with medium or perfusion of medium through the intact vasculature. Late follicular development, ovulation, and steroidogenesis can also be examined in these systems. Another approach has been to culture individual follicles isolated by enzymatic or mechanical dissociation. The majority of this research has focused on improving follicular development in vitro. This review will discuss these various culture techniques and some of the results that have been acquired. Recent results from toxicological studies utilizing whole ovarian cultures performed will also be described. Future applications for ovarian and follicular cultures may include in vitro follicular development for eventual production of offspring from frozen ovarian tissue, mechanistic studies related to the impact of endocrine disruptors and ovotoxicants on ovarian function, and further investigations into follicle activation and development. PMID:12161346

  20. Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

    NASA Astrophysics Data System (ADS)

    Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

    2014-03-01

    Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

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

    PubMed Central

    Peel, Trisha N.; Dylla, Brenda L.; Hughes, John G.; Lynch, David T.; Greenwood-Quaintance, Kerryl E.; Cheng, Allen C.; Mandrekar, Jayawant N.

    2016-01-01

    ABSTRACT 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. PMID:26733067

  2. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cell and tissue culture supplies and equipment... 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...

  3. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and equipment... 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...

  4. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cell and tissue culture supplies and equipment... 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...

  5. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cell and tissue culture supplies and equipment... 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...

  6. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cell and tissue culture supplies and equipment... 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...

  7. Mineralization and growth of cultured embryonic skeletal tissue in microgravity

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

    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.

  8. Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

    PubMed Central

    Ng-Sui-Hing, Ng-Kwet-Leok A.; Rabe, Brian A.; Lewis, Brittany B.; Saha, Margaret S.

    2012-01-01

    The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18. While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45. Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of

  9. Dissection, culture, and analysis of Xenopus laevis embryonic retinal tissue.

    PubMed

    McDonough, Molly J; Allen, Chelsea E; Ng-Sui-Hing, Ng-Kwet-Leok A; Rabe, Brian A; Lewis, Brittany B; Saha, Margaret S

    2012-01-01

    The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates (12,14-18). While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells (7,19-23). For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues (8,19-22,24-33). Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level (5,8,21,24,27-30,33-39). Xenopus laevis, a classic model system for the study of early neural development (19,27,29,31-32,40-42), serves as a particularly suitable system for retinal primary cell culture (10,38,43-45). Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction (25,38,43). In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding

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

    SciTech Connect

    Sahai, O.; Knuth, M.

    1985-03-01

    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.

  11. Effects of cold atmospheric plasma on mucosal tissue culture

    NASA Astrophysics Data System (ADS)

    Welz, Christian; Becker, Sven; Li, Yang-Fang; Shimizu, Tetsuji; Jeon, Jin; Schwenk-Zieger, Sabina; Thomas, Hubertus M.; Isbary, Georg; Morfill, Gregor E.; Harréus, Ulrich; Zimmermann, Julia L.

    2013-01-01

    Thermal plasmas have been commonly used in medical applications such as plasma ablation and blood coagulation. Newer developments show that plasmas can be generated with ion temperatures close to room temperature: these non-thermal or so-called cold atmospheric plasmas (CAPs) therefore open up a wide range of further biomedical applications. Based on the understanding of the bactericidal, virucidal and fungicidal properties of CAPs, information about the effects of CAP on mucosal cells and tissue is still lacking. Therefore this study focuses on the interaction of CAP with healthy head and neck mucosal cells on a molecular level. To analyse this interaction in detail, fresh tissue samples from healthy nasal and pharyngeal mucosa were harvested during surgery, assembled to a three-dimensional tissue culture model (mini organ cultures) and treated with CAP for different treatment times. Effects on the viability, necrosis induction and mutagenic activity were evaluated with the trypan blue exclusion test, Annexin-V/PI staining and alkaline microgel electrophoresis (comet assay). Trypan blue exclusion test revealed that the CAP treatment significantly decreases the cell viability for all tested treatment times (5, 10, 30, 60 and 120 s p < 0.05), but only a treatment time of 120 s showed a cytotoxic effect as the viability dropped below 90%. Annexin-V/PI staining revealed a significant increase in necrosis in CAP treated pharyngeal tissue cultures for treatment times of 60 and 120 s (p < 0.05). For nasal tissue this effect was already detected for a 30 s treatment (p < 0.05). Comet assay analysis showed no mutagenic effects after exposure to CAP.

  12. Hydrodynamic effects on cells in agitated tissue culture reactors

    NASA Technical Reports Server (NTRS)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  13. Micropropagation and tissue culture of Eucalyptus-a review.

    PubMed

    Le Roux, J J; Staden, J V

    1991-12-01

    Micropropagation has the potential to provide very high multiplication rates of selected tree genotypes, with resulting short-term silvicultural gains. Aseptic cultures have been established from seeds, seedlings, shoots, flowers and lignotubers. Callus cultures have been established from a wide range of tissue sources for at least 30 species of Eucalyptus. Plant regeneration from callus was successful for 12 of these species. Micropropagation through axillary proliferation, or adventitious shoot proliferation on nodal explants, or both, has been successful. An agar-based medium of Murashige and Skoog with a low auxin/cytokinin ratio is most commonly used for shoot multiplication. Vitrification and shoot senescence remain problems. Gibberellic acid was added in some media to stimulate shoot elongation. Various media are used for in vitro root initiation. Suspension and protoplast cultures have been achieved and plants have been regenerated from protoplasts. In vitro techniques are presently being applied to Eucalyptus to achieve genetic transformations. PMID:14972839

  14. Lemongrass-Incorporated Tissue Conditioner Against Candida albicans Culture

    PubMed Central

    Amornvit, Pokpong; Srithavaj, Theerathavaj

    2014-01-01

    Background: Tissue conditioner is applied popularly with dental prosthesis during wound healing process but it becomes a reservoir of oral microbiota, especially Candida species after long-term usage. Several antifungal drugs have been mixed with this material to control fungal level. In this study, lemongrass essential oil was added into COE-COMFORT tissue conditioner before being determined for anti-Candida efficacy. Materials and Methods: Lemongrass (Cymbopogon citratus) essential oil was primarily determined for antifungal activity against C. albicans American type culture collection (ATCC) 10231 and MIC (minimum inhibitory concentration) value by agar disk diffusion and broth microdilution methods, respectively. COE-COMFORT tissue conditioner was prepared as recommended by the manufacturer after a fixed volume of the oil at its MIC or higher concentrations were mixed thoroughly in its liquid part. Antifungal efficacy of the tissue conditioner with/without herb was finally analyzed. Results: Lemongrass essential oil displayed potent antifungal activity against C. albicans ATCC 10231and its MIC value was 0.06% (v/v). Dissimilarly, the tissue conditioner containing the oil at MIC level did not cease the growth of the tested fungus. Both reference and clinical isolates of C. albicans were completely inhibited after exposed to the tissue conditioner containing at least 0.25% (v/v) of the oil (approximately 4-time MIC). The tissue conditioner without herb or with nystatin was employed as negative or positive control, respectively. Conclusion: COE-COMFORT tissue conditioner supplemented with lemongrass essential oil obviously demonstrated another desirable property as in vitro anti-Candida efficacy to minimize the risk of getting Candidal infection. PMID:25177638

  15. Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.

    PubMed

    Kliot, Adi; Kontsedalov, Svetlana; Lebedev, Galina; Brumin, Marina; Cathrin, Pakkianathan Britto; Marubayashi, Julio Massaharu; Skaljac, Marisa; Belausov, Eduard; Czosnek, Henryk; Ghanim, Murad

    2014-01-01

    Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell. PMID:24637389

  16. Design of a Miniature Tissue Culture System to Culture Mouse Heart Valves

    PubMed Central

    Lieber, Samuel C.; Kruithof, Boudewijn P. T.; Aubry, Nadine; Vatner, Stephen F.; Gaussin, Vinciane

    2010-01-01

    Valvular heart disease is a leading cause of morbidity and mortality in adults but little is known about the underlying etiology. A better understanding of the genetic and hemodynamic mechanisms involved in growth and remodeling of heart valves during physiological and pathological conditions is needed for a better understanding of valvular heart disease. Here, we report the design of a miniature tissue culture system (MTCS) that allows the culture of mitral valves from perinatal to adult mice. The design of the MTCS is novel in that fine positioning and cannulation can be conducted with hearts of different sizes (perinatal to adult). Perfusion of the heart and hence, culture of the mitral valve in its natural position, occurs in a hydraulically sealed culture bath environment. Using the MTCS, we successfully cultured the mitral valve of adult mouse hearts for 3 days. Histological analysis indicated that the cultured valves remained viable and their extracellular matrix organization was similar to age-matched native valves. Gene expression could also be modified in cultured valves by perfusion with medium containing beta-galactosidase-expressing adenovirus. Thus, the MTCS is a new tool to study the genetic and hemodynamic mechanisms underlying the three-dimensional organization of the heart valves, which could provide insights in the pathology of valvular heart disease and be used in animal models for the development of tissue-engineered heart valves. PMID:20099034

  17. Noninvasive Oxygen Monitoring in Three-Dimensional Tissue Cultures Under Static and Dynamic Culture Conditions

    PubMed Central

    Weyand, Birgit; Nöhre, Mariel; Schmälzlin, Elmar; Stolz, Marvin; Israelowitz, Meir; Gille, Christoph; von Schroeder, Herb P.; Reimers, Kerstin; Vogt, Peter M.

    2015-01-01

    Abstract We present a new method for noninvasive real-time oxygen measurement inside three-dimensional tissue-engineered cell constructs in static and dynamic culture settings in a laminar flow bioreactor. The OPAL system (optical oxygen measurement system) determines the oxygen-dependent phosphorescence lifetime of spherical microprobes and uses a two-frequency phase-modulation technique, which fades out the interference of background fluorescence from the cell carrier and culture medium. Higher cell densities in the centrum of the scaffolds correlated with lower values of oxygen concentration obtained with the OPAL system. When scaffolds were placed in the bioreactor, higher oxygen values were measured compared to statically cultured scaffolds in a Petri dish, which were significantly different at day 1–3 of culture. This technique allows the use of signal-weak microprobes in biological environments and monitors the culture process inside a bioreactor. PMID:26309802

  18. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Synthetic cell and tissue culture media and... 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....

  19. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and... 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....

  20. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Synthetic cell and tissue culture media and... 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....

  1. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Synthetic cell and tissue culture media and... 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....

  2. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Synthetic cell and tissue culture media and... 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....

  3. Defined media for plant tissue culture: Final report

    SciTech Connect

    Good, N.E.

    1986-01-01

    This grant was for the purpose of developing improved plant tissue culture media. The rationale was to introduce the use of low pKa hydrogen ion buffers to stabilize pH and to introduce the use of slow release forms of the plant hormones, auxin and cytokinin, to provide the tissues with a constant supply of these essential factors. The zwittionic buffer, MES, proved useful for pH stabilization, while a wide range of indoleacetylamino acids provided a wide range of levels of available amino acids with consequent different levels of development of shoots, roots or callus. In general, some free indoleacetic acid in addition to the conjugate seemed necessary for organogenesis, but this phenomenon depended very much on the level of cytokinin. Time did not permit us to make any significant progress in the development of slow-release forms of cytokinins. 2 figs.

  4. Anaerobic cultures from preserved tissues of baby mammoth

    NASA Astrophysics Data System (ADS)

    Pikuta, Elena V.; Fisher, Daniel; Hoover, Richard B.

    2011-10-01

    Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 3 oC. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that keeps other bacteria from colonizing a system. Permafrost and lactic acid preserved the body of this one month-old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete sample of the species ever recovered. The diversity of novel psychrophilic anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here, we discuss the specifics of the isolation of new psychrophilic strains, differentiation from trivial contamination, and preliminary results for characterization of the cultures.

  5. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    PubMed

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines. PMID:27613045

  6. Anaerobic Cultures from Preserved Tissues of Baby Mammoth

    NASA Technical Reports Server (NTRS)

    Pikuta, Elena V.; Hoover, Richard B.; Fisher, Daniel

    2011-01-01

    Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 4 C. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that prevents other bacteria from over-dominating a system. Permafrost and lactic acid preserved the body of this one-month old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete such specimen ever recovered. The diversity of novel anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here we discuss the specifics of the isolation of new strains, differentiation from trivial contamination, and preliminary results for the characterization of cultures.

  7. Chitinases and Imaginal disc growth factors organize the extracellular matrix formation at barrier tissues in insects

    PubMed Central

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Patil, Kapil R; Loch, Gerrit; Behr, Matthias

    2016-01-01

    The cuticle forms an apical extracellular-matrix (ECM) that covers exposed organs, such as epidermis, trachea and gut, for organizing morphogenesis and protection of insects. Recently, we reported that cuticle proteins and chitin are involved in ECM formation. However, molecular mechanisms that control assembly, maturation and replacement of the ECM and its components are not well known. Here we investigated the poorly described glyco-18-domain hydrolase family in Drosophila and identified the Chitinases (Chts) and imaginal-disc-growth-factors (Idgfs) that are essential for larval and adult molting. We demonstrate that Cht and idgf depletion results in deformed cuticles, larval and adult molting defects, and insufficient protection against wounding and bacterial infection, which altogether leads to early lethality. We show that Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/idgf4/idgf5/idgf6 are needed for organizing proteins and chitin-matrix at the apical cell surface. Our data indicate that normal ECM formation requires Chts, which potentially hydrolyze chitin-polymers. We further suggest that the non-enzymatic idgfs act as structural proteins to maintain the ECM scaffold against chitinolytic degradation. Conservation of Chts and Idgfs proposes analogous roles in ECM dynamics across the insect taxa, indicating that Chts/Idgfs are new targets for species specific pest control. PMID:26838602

  8. Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    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.

  9. Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    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.

  10. An inexpensive portable incubator for tissue or cell cultures.

    PubMed

    Mealing, G A; Schwartz, J L

    1989-01-01

    A lightweight incubator is described which is suitable for transporting cells or tissues maintained in Petri dishes. Since individual cultures are held in a sealed environment, sterility of samples is maintained, and pH remains stable for extended periods, even when bicarbonate buffered media is used. The incubator is battery powered, or may be connected to an automotive electrical power source. It has been designed to minimize exposure of the samples to mechanical shock. The components required for construction are inexpensive. PMID:2804706

  11. Identification of Eucalyptus citriodora clones micropropagated in tissue culture.

    PubMed

    Kojima, E; Izumi, M; Tanabe, T; Matsuda, M; Shimizu, T; Murakami, A; Murakami, K

    1997-01-01

    The extent of genetic identity observed in the young individuals which were micropropagated from a single Eucalyptus individual was analyzed by using DNA-fingerprinting. Among 40,000 tissue-cultured-seedings of E.citriodora, 200 plants were randomly chosen so that each total DNA might be extracted from their leaves. Using these DNAs as template, PCR was performed with some primers we found in advance that leads polymorphism for DNA of E. citriodora. In this study, all over the 200 cases, the band pattern formed cDNA fragment on a gel after electrophoresis was the identical one mutually. PMID:9586054

  12. Gradients of metabolite accumulation and redifferentiation of nutritive cells associated with vascular tissues in galls induced by sucking insects.

    PubMed

    Carneiro, Renê Gonçalves da Silva; Isaias, Rosy Mary Dos Santos

    2015-01-01

    Plant cells respond to abiotic and biotic stimuli, which generate adaptive phenotypes in plant organs. In the case of plant galls, cell phenotypes are adaptive for the gall inducer and assume characteristics mainly linked to its protection and nutrition. Herein, the cytological development and histochemical profile of Nothotrioza cattleiani galls, a sucking insect, on the leaves of Psidium cattleianum are compared with those of other galls, especially N. myrtoidis galls, searching for conserved and divergent alterations in cell fates and cycles. Leaf cell fates are completely changed within galls, except for epidermal cells, but the comparison between Nothotrioza spp. galls shows conserved fates. Nevertheless, cytological development of N. cattleiani galls is different from the standby-redifferentiation of N. myrtoidis galls. Starch and lignins, and reducing sugars form centrifugal and centripetal gradients of accumulation, respectively. Proteins, total phenolics, terpenoids, proanthocyanidins and reactive oxygen species are detected in bidirectional gradients, i.e. weak or undetectable reaction in the median cortical cells that is gradually more intense in the cell layers towards the inner and outer surfaces of the gall. True nutritive cells associated with vascular tissues, together with the bidirectional gradients of metabolite accumulation, are herein reported for the first time in insect galls. The globoid galls of N. cattleiani, though macro-morphologically similar to the galls of N. myrtoidis, are distinct and unique among insect galls, as far as the cellular, subcellular and histochemical traits are concerned. Thus, the traits of the galls on P. cattleianum studied herein represent the extended phenotypes of their inducers. PMID:26209687

  13. Gradients of metabolite accumulation and redifferentiation of nutritive cells associated with vascular tissues in galls induced by sucking insects

    PubMed Central

    Carneiro, Renê Gonçalves da Silva; Isaias, Rosy Mary dos Santos

    2015-01-01

    Plant cells respond to abiotic and biotic stimuli, which generate adaptive phenotypes in plant organs. In the case of plant galls, cell phenotypes are adaptive for the gall inducer and assume characteristics mainly linked to its protection and nutrition. Herein, the cytological development and histochemical profile of Nothotrioza cattleiani galls, a sucking insect, on the leaves of Psidium cattleianum are compared with those of other galls, especially N. myrtoidis galls, searching for conserved and divergent alterations in cell fates and cycles. Leaf cell fates are completely changed within galls, except for epidermal cells, but the comparison between Nothotrioza spp. galls shows conserved fates. Nevertheless, cytological development of N. cattleiani galls is different from the standby-redifferentiation of N. myrtoidis galls. Starch and lignins, and reducing sugars form centrifugal and centripetal gradients of accumulation, respectively. Proteins, total phenolics, terpenoids, proanthocyanidins and reactive oxygen species are detected in bidirectional gradients, i.e. weak or undetectable reaction in the median cortical cells that is gradually more intense in the cell layers towards the inner and outer surfaces of the gall. True nutritive cells associated with vascular tissues, together with the bidirectional gradients of metabolite accumulation, are herein reported for the first time in insect galls. The globoid galls of N. cattleiani, though macro-morphologically similar to the galls of N. myrtoidis, are distinct and unique among insect galls, as far as the cellular, subcellular and histochemical traits are concerned. Thus, the traits of the galls on P. cattleianum studied herein represent the extended phenotypes of their inducers. PMID:26209687

  14. Functional interpretation of a non-gut hemocoelic tissue aminopeptidase N (APN) in a lepidopteran insect pest Achaea janata.

    PubMed

    Ningshen, Thuirei Jacob; Aparoy, Polamarasetty; Ventaku, Venkat Rao; Dutta-Gupta, Aparna

    2013-01-01

    Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn(++) dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation. PMID:24244508

  15. Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

    PubMed Central

    Ningshen, Thuirei Jacob; Aparoy, Polamarasetty; Ventaku, Venkat Rao; Dutta-Gupta, Aparna

    2013-01-01

    Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn++ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation. PMID:24244508

  16. Fluorescent in situ hybridization for the localization of viruses, bacteria and other microorganisms in insect and plant tissues.

    PubMed

    Kliot, Adi; Ghanim, Murad

    2016-04-01

    Methods for the localization of cellular components such as nucleic acids, proteins, cellular vesicles and more, and the localization of microorganisms including viruses, bacteria and fungi have become an important part of any research program in biological sciences that enable the visualization of these components in fixed and live tissues without the need for complex processing steps. The rapid development of microscopy tools and technologies as well as related fluorescent markers and fluorophores for many cellular components, and the ability to design DNA and RNA sequence-based molecular probes and antibodies which can be visualized fluorescently, have rapidly advanced this field. This review will focus on some of the localizations methods which have been used in plants and insect pests in agriculture, and other microorganisms, which are rapidly advancing the research in agriculture-related fields. PMID:26678796

  17. Critical tissue residue approach linking accumulated metals in aquatic insects to population and community-level effects

    USGS Publications Warehouse

    Schmidt, Travis S.; Clements, William H.; Zuellig, Robert E.; Mitchell, Katharine A.; Church, Stanley E.; Wanty, Richard B.; San Juan, Carma A.; Adams, Monique; Lamothe, Paul J.

    2011-01-01

    Whole body Zn concentrations in individuals (n = 825) from three aquatic insect taxa (mayflies Rhithrogena spp. and Drunella spp. and the caddisfly Arctopsyche grandis) were used to predict effects on populations and communities (n = 149 samples). Both mayflies accumulated significantly more Zn than the caddisfly. The presence/absence of Drunella spp. most reliably distinguished sites with low and high Zn concentrations; however, population densities of mayflies were more sensitive to increases in accumulated Zn. Critical tissue residues (634 (mu or u)g/g Zn for Drunella spp. and 267 (mu or u)g/g Zn for Rhithrogena spp.) caused a 20% reduction in maximum (90th quantile) mayfly densities. These critical tissue residues were associated with exposure to 7.0 and 3.9 (mu or u)g/L dissolved Zn for Drunella spp. and Rhithrogena spp., respectively. A threshold in a measure of taxonomic completeness (observed/expected) was observed at 5.4 (mu or u)g/L dissolved Zn. Dissolved Zn concentrations associated with critical tissue residues in mayflies were also associated with adverse effects in the aquatic community as a whole. These effects on populations and communities occurred at Zn concentrations below the U.S. EPA hardness-adjusted continuous chronic criterion.

  18. Physiological properties of vertebrate nerve cells in tissue culture.

    PubMed

    Dichter, M A

    1975-01-01

    Vertebrate neurons in tissue culture are providing us with a new model system for studying the complex events which occur during neuronal differentiation, synaptogenesis, and neural network formation. It is already apparent that dissociated embryo neurons are capable of differentiating both morphologically and physiologically along predetermined lines in the absence of external influences. These neurons can form new connections with one another but retain some specificity in their selections. Both simple and complex neural networks can be seen. At the present time, the development of the invitro model system is just being explored. The potential value of a system of this kind at a variety of investigative levels should be appreciated. Questions of a fundamental nature in neurobiology, such as how synapses form, what rules govern such interaction, how cells recognize one another, and the nature of the basic two-, three-, or four-cell circuits that comprise the more complex neurons tissue can be approached with this system. Studies of the neurons and synapses themselves can lead to a more basic understanding of vertebrate nervous system functioning. The development of certain pathophysiological processes and the effects of neuroactive drugs on vertebrate neurons may be studied at the cellular level. Finally, the basic mechanism of some genetic abnormalities which produce abnormal nervous structure and function may be more easily determined in a simplified in vitro model than in the intact central nervous system. The value of any model is not inherent in the elegance of the model itseld, but only in its ability to suggest answers to fundamental questions about the system being modeled. Many fundamental questions about brain mechanisms in mental retardation remain unanswered. Perhaps some day the model of nerve cells in tissue culture will bring us closer to the answers to these questions. PMID:173059

  19. Curvature-dependent excitation propagation in cultured cardiac tissue

    NASA Astrophysics Data System (ADS)

    Kadota, S.; Kay, M. W.; Magome, N.; Agladze, K.

    2012-02-01

    The geometry of excitation wave front may play an important role on the propagation block and spiral wave formation. The wave front which is bent over the critical value due to interaction with the obstacles may partially cease to propagate and appearing wave breaks evolve into rotating waves or reentry. This scenario may explain how reentry spontaneously originates in a heart. We studied highly curved excitation wave fronts in the cardiac tissue culture and found that in the conditions of normal, non-inhibited excitability the curvature effects do not play essential role in the propagation. Neither narrow isthmuses nor sharp corners of the obstacles, being classical objects for production of extremely curved wave front, affect non-inhibited wave propagation. The curvature-related phenomena of the propagation block and wave detachment from the obstacle boundary were observed only after partial suppression of the sodium channels with Lidocaine. Computer simulations confirmed the experimental observations. The explanation of the observed phenomena refers to the fact that the heart tissue is made of finite size cells so that curvature radii smaller than the cardiomyocyte size loses sense, and in non-inhibited tissue the single cell is capable to transmit excitation to its neighbors.

  20. Metabolic measurements in cell culture and tissue constructs

    NASA Astrophysics Data System (ADS)

    Rolfe, P.

    2008-10-01

    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#

  1. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

    PubMed Central

    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.

    2014-01-01

    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, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D 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), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 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. PMID:24859528

  2. Increased Plasminogen Activator (Urokinase) in Tissue Culture After Fibrin Deposition

    PubMed Central

    Bernik, Maria B.

    1973-01-01

    Lysis of fibrin in tissue culture has been shown to be due to plasminogen activator identified immunologically as urokinase. The present study examines fibrinolytic events in culture, particularly mechanisms leading to increased urokinase levels and accelerated fibrinolysis. Deposition of fibrin on cells in culture was followed by a two- to six-fold increase in urokinase in the supernates and rapid disappearance of the fibrin. Investigation of factors that might be responsible for these events (including fibrin, fibrinogen, vasoactive stimuli, and the enzymes thrombin and plasmin) indicated that the enhanced urokinase yields were mediated through plasmin and thrombin. Study of the possible modes of action of thrombin and plasmin indicated that these enzymes are capable of acting on the cells themselves as well as on cell-produced material. The effect on cells was manifested by mitotic activity or, occasionally, cell injury and death. Although these effects influenced urokinase levels, enhanced yields were explained best by the action of enzymes on cellproduced material. Studies with plasmin and thrombin, and also trypsin, indicated that proteolytic enzymes may act in various ways—affect the stability of urokinase, interfere with inhibition of urokinase by naturally occurring inhibitor(s), and induce urokinase activity from inactive material. Plasma and thrombin appeared to act primarily through the latter mechanism. Inactive material, which gave rise to urokinase upon exposure to proteolytic enzymes and which may represent urokinase precursor, was found in cultures of kidney, lung, spleen, and thyroid. Urokinase in such inactive state appears to be readily accessible to activation by enzymes, particularly plasmin and thrombin, thus facilitating removal of fibrin and possibly also providing pathways to excessive fibrinolysis. PMID:4266421

  3. Lipid composition of slash pine tissue cultures grown with lunar and earth soils

    NASA Technical Reports Server (NTRS)

    Laseter, J. L.; Weete, J. D.; Baur, P. S.; Walkinshaw, C. H.

    1973-01-01

    Lipid analyses were conducted on slash pine tissues grown in culture in the presence of lunar (Apollo 15) and earth soils. Significant reductions in the total lipids, fatty acids, and sterol components were found in the tissues grown in contact with each of the soils employed when compared to the control. Tissues grown with lunar soil showed the greatest reductions. These results are discussed with respect to previous ultrastructural studies on similarly treated slash pine tissues and lipid analyses on tobacco tissue cultures.

  4. Organoid culture systems for prostate epithelial tissue and prostate cancer tissue

    PubMed Central

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

    2016-01-01

    Summary This protocol describes a recently developed strategy to generate 3D prostate organoid cultures from healthy mouse and human prostate (either bulk or FAC-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumour 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 tumour. The stepwise establishment of these cultures and the fully defined serum-free conditioned medium that is required to sustain organoid growth are outlined. Organoids established using this protocol can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis and drug discovery. PMID:26797458

  5. Flexible automation of cell culture and tissue engineering tasks.

    PubMed

    Knoll, Alois; Scherer, Torsten; Poggendorf, Iris; Lütkemeyer, Dirk; Lehmann, Jürgen

    2004-01-01

    Until now, the predominant use cases of industrial robots have been routine handling tasks in the automotive industry. In biotechnology and tissue engineering, in contrast, only very few tasks have been automated with robots. New developments in robot platform and robot sensor technology, however, make it possible to automate plants that largely depend on human interaction with the production process, e.g., for material and cell culture fluid handling, transportation, operation of equipment, and maintenance. In this paper we present a robot system that lends itself to automating routine tasks in biotechnology but also has the potential to automate other production facilities that are similar in process structure. After motivating the design goals, we describe the system and its operation, illustrate sample runs, and give an assessment of the advantages. We conclude this paper by giving an outlook on possible further developments. PMID:15575718

  6. Methods for the Organogenesis of Skeletal Muscle in Tissue Culture

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman; Shansky, Janet; DelTatto, Michael; Chromiak, Joseph

    1997-01-01

    Skeletal muscle structure is regulated by many factors, including nutrition, hormones, electrical activity, and tension. The muscle cells are subjected to both passive and active mechanical forces at all stages of development and these forces play important but poorly understood roles in regulating muscle organogenesis and growth. For example, during embryogenesis, the rapidly growing skeleton places large passive mechanical forces on the attached muscle tissue. These forces not only help to organize the proliferating mononucleated myoblasts into the oriented, multinucleated myofibers of a functional muscle but also tightly couple the growth rate of muscle to that of bone. Postnatally, the actively contracting, innervated muscle fibers are subjected to different patterns of active and passive tensions which regulate longitudinal and cross sectional myofiber growth. These mechanically-induced organogenic processes have been difficult to study under normal tissue culture conditions, resulting in the development of numerous methods and specialized equipment to simulate the in vivo mechanical environment.These techniques have led to the "engineering" of bioartificial muscles (organoids) which display many of the characteristics of in vivo muscle including parallel arrays of postmitotic fibers organized into fascicle-like structures with tendon-like ends. They are contractile, express adult isoforms of contractile proteins, perform directed work, and can be maintained in culture for long periods. The in vivo-like characteristics and durability of these muscle organoids make them useful for long term in vitro studies on mechanotransduction mechanisms and on muscle atrophy induced by decreased tension. In this report, we described a simple method for generating muscle organoids from either primary embrionic avain or neonatal rodent myoblasts.

  7. Adipose Tissue Engineering in Three-Dimensional Levitation Tissue Culture System Based on Magnetic Nanoparticles

    PubMed Central

    Daquinag, Alexes C.; Souza, Glauco R.

    2013-01-01

    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

  8. Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.

    PubMed

    Jones, Samuel L; Valenzisi, Amy; Sontakke, Sushama; Sprayberry, Kimberly A; Maggi, Ricardo; Hegarty, Barbara; Breitschwerdt, Edward

    2007-03-31

    Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid. PMID:17204376

  9. Dynamics of Necrophagous Insect and Tissue Bacteria for Postmortem Interval Estimation During the Warm Season in Romania.

    PubMed

    Iancu, Lavinia; Sahlean, Tiberiu; Purcarea, Cristina

    2016-01-01

    The estimation of postmortem interval (PMI) is affected by several factors including the cause of death, the place where the body lay after death, and the weather conditions during decomposition. Given the climatic differences among biogeographic locations, the understanding of necrophagous insect species biology and ecology is required when estimating PMI. The current experimental model was developed in Romania during the warm season in an outdoor location. The aim of the study was to identify the necrophagous insect species diversity and dynamics, and to detect the bacterial species present during decomposition in order to determine if their presence or incidence timing could be useful to estimate PMI. The decomposition process of domestic swine carcasses was monitored throughout a 14-wk period (10 July-10 October 2013), along with a daily record of meteorological parameters. The chronological succession of necrophagous entomofauna comprised nine Diptera species, with the dominant presence of Chrysomya albiceps (Wiedemann 1819) (Calliphoridae), while only two Coleoptera species were identified, Dermestes undulatus (L. 1758) and Creophilus maxillosus Brahm 1970. The bacterial diversity and dynamics from the mouth and rectum tissues, and third-instar dipteran larvae were identified using denaturing gradient gel electrophoresis analysis and sequencing of bacterial 16S rRNA gene fragments. Throughout the decomposition process, two main bacterial chronological groups were differentiated, represented by Firmicutes and Gammaproteobacteria. Twenty-six taxa from the rectal cavity and 22 from the mouth cavity were identified, with the dominant phylum in both these cavities corresponding to Firmicutes. The present data strengthen the postmortem entomological and microbial information for the warm season in this temperate-continental area, as well as the role of microbes in carcass decomposition. PMID:26487246

  10. Tissue-specific promoter usage and diverse splicing variants of found in neurons, an ancestral Hu/ELAV-like RNA-binding protein gene of insects, in the direct-developing insect Gryllus bimaculatus.

    PubMed

    Watanabe, T; Aonuma, H

    2014-02-01

    Hu/ELAV-like RNA-binding proteins (RBPs) are involved in the post-transcriptional regulation of RNA metabolism including splicing, transport, translational control and turnover. The Hu/ELAV-like RBP genes are predominantly expressed in neurons, and are therefore used as common neuronal markers in many animals. Although the expression patterns and functions of the Hu/ELAV-like RBP genes have been extensively studied in the model insect Drosophila melanogaster, little is known in basal direct-developing insects. In the present study, we performed an identification and expression analysis of the found in neurons (fne) gene, an ancestral insect Hu/ELAV-like RBP gene, in the cricket Gryllus bimaculatus. Contrary to expectation that the Gryllus fne transcript would be predominantly expressed in the nervous system, expression analysis revealed that the Gryllus fne gene is expressed broadly. In addition, we discovered that alternative promoter usage directs tissue-specific and embryonic stage-dependent regulation of fne expression, and that alternative splicing contributes to the generation of diverse sets of fne transcripts. Our data provide novel insights into the evolutionary diversification of the Hu/ELAV-like RBP gene family in insects. PMID:24382152

  11. Effects of Apollo 12 lunar material on lipid levels of tobacco tissue and slash pine cultures

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1972-01-01

    Investigations of the lipid components of pine tissues (Pinus elloitii) are discussed, emphasizing fatty acids and steroids. The response by slash pine tissue cultures to growth in contact with Apollo lunar soil, earth basalt, and Iowa soil is studied. Tissue cultures of tobacco grown for 12 weeks in contact with lunar material from Apollo 12 flight contained 21 to 35 percent more total pigment than control tissues. No differences were noted in the fresh or dry weight of the experimental and control samples.

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

    PubMed

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

    2015-05-01

    Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. PMID:24493315

  13. Gastroenterology-urology devices; classification of tissue culture media for human ex vivo tissue and cell culture processing applications. Final rule.

    PubMed

    2001-05-16

    The Food and Drug Administration (FDA) is classifying tissue culture media for human ex vivo tissue and cell culture processing applications into class II (special controls). The special control that will apply to this device is a guidance document entitled "Class II Special Controls Guidance Document: issue Culture Media for Human Ex Vivo Tissue and Cell Culture Processing Applications; Final Guidance for Industry and FDA Reviewers." The agency is taking this action in response to a petition submitted under the Federal Food, Drug, and Cosmetic Act (the act) as amended by the Medical Device Amendments of 1976, the Safe Medical Devices Act of 1990, and the Food and Drug Administration Modernization Act of 1997. The agency is classifying these devices into class II (special controls) in order to provide a reasonable assurance of the safety and effectiveness of the devices. PMID:11721690

  14. Pre-metatarsal skeletal development in tissue culture at unit- and microgravity

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

    Explant organ culture was used to demonstrate that isolated embryonic mouse pre-metatarsal mesenchyme is capable of undergoing a series of differentiative and morphogenetic developmental events. Mesenchyme differentiation into chondrocytes, and concurrent morphogenetic patterning of the cartilage tissue, and terminal chondrocyte differentiation with subsequent matrix mineralization show that cultured tissue closely parallels in vivo development. Whole mount alizarin red staining of the cultured tissue demonstrates that the extracellular matrix around the hypertrophied chondrocytes is competent to support mineralization. Intensely stained mineralized bands are similar to those formed in pre-metatarsals developing in vivo. We have adapted the culture strategy for experimentation in a reduced gravity environment on the Space Shuttle. Spaceflight culture of pre-metatarsals, which have already initiated chondrogenesis and morphogenetic patterning, results in an increase in cartilage rod size and maintenance of rod shape, compared to controls. Older pre-metatarsal tissue, already terminally differentiated to hypertrophied cartilage, maintained rod structure and cartilage phenotype during spaceflight culture.

  15. Biosynthesis of Sterols and Triterpenoids in Tissue Cultures of Cucurbita maxima.

    PubMed

    Caputo, O; Delprino, L; Viola, F; Caramiello, R; Balliano, G

    1983-11-01

    The biosynthesis of sterols and triterpenoids in CUCURBITA MAXIMA was studied by analysis of unsaponifiable fraction of tissues from different development stages of the plant (seeds, seedlings, adult plant and tissue culture) and by feeding germinating seeds and tissue cultures with [2- (14)C]-acetate. Synthesis of cucurbitacins does not occur in callus tissues of CUCURBITA MAXIMA, whereas a wide variety of 4,4-dimethylsterols present in these tissues testifies of a high level of squaleneoxide cyclase activity in growing callus. The peculiarity of Cucurbitaceae among the higher plants is also discussed comparing the side chain biosynthesis of sterols in CUCURBITA MAXIMA to that operating in other higher plants. PMID:17405044

  16. Inducibility of chemical defences by two chewing insect herbivores in pine trees is specific to targeted plant tissue, particular herbivore and defensive trait.

    PubMed

    Moreira, Xoaquín; Lundborg, Lina; Zas, Rafael; Carrillo-Gavilán, Amparo; Borg-Karlson, Anna-Karin; Sampedro, Luis

    2013-10-01

    There is increasing evidence that plants can react to biotic aggressions with highly specific responses. However, few studies have attempted to jointly investigate whether the induction of plant defences is specific to a targeted plant tissue, plant species, herbivore identity, and defensive trait. Here we studied those factors contributing to the specificity of induced defensive responses in two economically important pine species against two chewing insect pest herbivores. Juvenile trees of Pinus pinaster and P. radiata were exposed to herbivory by two major pest threats, the large pine weevil Hylobius abietis (a bark-feeder) and the pine processionary caterpillar Thaumetopoea pityocampa (a folivore). We quantified in two tissues (stem and needles) the constitutive (control plants) and herbivore-induced concentrations of total polyphenolics, volatile and non-volatile resin, as well as the profile of mono- and sesquiterpenes. Stem chewing by the pine weevil increased concentrations of non-volatile resin, volatile monoterpenes, and (marginally) polyphenolics in stem tissues. Weevil feeding also increased the concentration of non-volatile resin and decreased polyphenolics in the needle tissues. Folivory by the caterpillar had no major effects on needle defensive chemistry, but a strong increase in the concentration of polyphenolics in the stem. Interestingly, we found similar patterns for all these above-reported effects in both pine species. These results offer convincing evidence that induced defences are highly specific and may vary depending on the targeted plant tissue, the insect herbivore causing the damage and the considered defensive compound. PMID:23768645

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

    PubMed

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

    2014-05-01

    Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. PMID:24602569

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

    SciTech Connect

    Sluis, C.

    1980-09-01

    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.

  19. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  20. Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels

    PubMed Central

    Mabry, Kelly M.; Payne, Samuel Z.; Anseth, Kristi S.

    2015-01-01

    Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011) [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled “Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype” (Mabry et al., 2015) [2]. PMID:26702427

  1. ORGAN CULTURE OF MID-FACIAL TISSUE AND SECONDARY PALATE

    EPA Science Inventory

    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...

  2. A modular culture system for the generation of multiple specialized tissues.

    PubMed

    Minuth, Will W; Denk, Lucia; Glashauser, Anne

    2010-04-01

    Numerous factors influence cell functions and tissue development in culture. A modular culture system has been developed to allow the control of many of these important environmental parameters. Optimal adhesion of cells is obtained by selecting an individual biomaterial. Selected specimens are mounted in a tissue carrier in order to protect it against damage during handling and after seeding cells, the carriers can be used in a series of compatible perfusion culture containers. This technique allows the simple bathing of growing tissue under continuous medium transport and the exposure of epithelia to a gradient with different fluids at the luminal and basal sides. A further container is made of transparent material to observe microscopically the developing tissue. In addition, a special model features a flexible silicone lid to apply force to mimic the mechanical load required for developing connective and muscular tissue. Perfusion culture of stem/progenitor cells at the interface of an artificial interstitium made by a polyester fleece results in the spatial development of tubules. During long term culture over weeks the growing tissue is continuously exposed to fresh nutrition and respiratory gas. The medium is transported in a constant flow or in pulses, preventing unstirred layers of fluid. A variety of applications of this modular system, described in this paper, demonstrates that the biological profile of cells and tissues can be strongly improved when perfusion culture with a permanent provision of fresh medium is applied. PMID:20096452

  3. A Well-Controlled Nucleus Pulposus Tissue Culture System with Injection Port for Evaluating Regenerative Therapies.

    PubMed

    Arkesteijn, Irene T M; Mouser, Vivian H M; Mwale, Fackson; van Dijk, Bart G M; Ito, Keita

    2016-05-01

    In vitro evaluation of nucleus pulposus (NP) tissue regeneration would be useful, but current systems for NP culture are not ideal for injections. The aim of this study was to develop a long-term culture system for NP tissue that allows injections of regenerative agents. Bovine caudal NPs were harvested and placed in the newly designed culture system. After equilibration of the tissue to 0.3 MPa the volume was fixed and the tissue was cultured for 28 days. The cell viability and extracellular matrix composition remained unchanged during the culture period and gene expression profiles were similar to those obtained in earlier studies. Furthermore, to test the responsiveness of bovine caudal NPs in the system, samples were cultured for 4 days and injected twice (day 1 and 3) with (1) PBS, (2) Link-N, for regeneration, and (3) TNF-α, for degeneration. It was shown that TNF-α increased COX2 gene expression, whereas no effect of Link-N was detected. In conclusion, the newly designed system allows long-term culture of NP tissue, wherein tissue reactions to injected stimulants can be observed. PMID:26294008

  4. Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

    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.

  5. A novel technique to overcome browning in tissue culture.

    PubMed

    Bhat, S R; Chandel, K P

    1991-09-01

    Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed. PMID:24221674

  6. Assessment of DNA methylation changes in tissue culture of Brassica napus.

    PubMed

    Gao, Y; Ran, L; Kong, Y; Jiang, J; Sokolov, V; Wang, Y

    2014-11-01

    Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue:cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rape-seed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture. PMID:25739287

  7. Therapeutically important proteins from in vitro plant tissue culture systems.

    PubMed

    Doran, Pauline M

    2013-01-01

    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. PMID:23210789

  8. How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture.

    ERIC Educational Resources Information Center

    Haldeman, Janice H.; Ellis, Jane P.

    1988-01-01

    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)

  9. Cloning higher plants from aseptically cultured tissues and cells

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1982-01-01

    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.

  10. Epithelial cell cultures from normal and cancerous human tissues.

    PubMed

    Owens, R B; Smith, H S; Nelson-Rees, W A; Springer, E L

    1976-04-01

    Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal. PMID:176412

  11. Dynamic quantitative phase images of pond life, insect wings, and in vitro cell cultures

    PubMed Central

    Creath, Katherine

    2011-01-01

    This paper presents images and data of live biological samples taken with a novel Linnik interference microscope. The specially designed optical system enables instantaneous and 3D video measurements of dynamic motions within and among live cells without the need for contrast agents. This “label-free”, vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with current magnifications of 10X (NA 0.3) and 20X (NA 0.5) and wavelengths of 660 nm and 785 nm over fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phase-measurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different mud puddle organisms such as paramecium, flagellates and rotifers will be presented, as will measurements of flying ant wings and cultures of human breast cancer cells. These data highlight examples of monitoring different biological processes and motions. The live presentation features 4D phase movies of these examples. PMID:24357900

  12. Dynamic quantitative phase images of pond life, insect wings, and in vitro cell cultures

    NASA Astrophysics Data System (ADS)

    Creath, Katherine

    2010-08-01

    This paper presents images and data of live biological samples taken with a novel Linnik interference microscope. The specially designed optical system enables instantaneous and 3D video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with current magnifications of 10X (NA 0.3) and 20X (NA 0.5) and wavelengths of 660 nm and 785 nm over fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phasemeasurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different mud puddle organisms such as paramecium, flagellates and rotifers will be presented, as will measurements of flying ant wings and cultures of human breast cancer cells. These data highlight examples of monitoring different biological processes and motions. The live presentation features 4D phase movies of these examples.

  13. Differential Expression Patterns in Chemosensory and Non-Chemosensory Tissues of Putative Chemosensory Genes Identified by Transcriptome Analysis of Insect Pest the Purple Stem Borer Sesamia inferens (Walker)

    PubMed Central

    Zhang, Ya-Nan; Jin, Jun-Yan; Jin, Rong; Xia, Yi-Han; Zhou, Jing-Jiang; Deng, Jian-Yu; Dong, Shuang-Lin

    2013-01-01

    Background A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. Methodology/Principal Findings We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. Conclusion Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other

  14. Stem tissue mass density is linked to growth and resistance to a stem-boring insect in Alternanthera philoxeroides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate how stem anatomical structure is linked to growth and resistance to stem-boring insects in a herbaceous species, six populations of alligatorweed (Alternanthera philoxeroides) were grown in a common garden. Stem growth rate (GR) of A. philoxeroides and pupation rate as an estimate of ...

  15. Saponins do not affect the ecdysteroid receptor complex but cause membrane permeation in insect culture cell lines.

    PubMed

    De Geyter, Ellen; Swevers, Luc; Soin, Thomas; Geelen, Danny; Smagghe, Guy

    2012-01-01

    This project studied the effects of four saponins with a triterpenoid (Quillajasaponaria saponin and aescin) or steroid structure (digitonin and diosgenin which is the deglycosylated form of dioscin) on insect cells, namely Schneider S2 cells of Drosophila melanogaster (Diptera). A series of different experiments were performed to investigate potential mechanisms of action by saponins with regard to ecdysteroid receptor (EcR) responsiveness, cell viability, cell membrane permeation, and induction of apoptosis with DNA fragmentation and caspase-3 like activity. Major results were that (1) exposure of S2 cells containing an EcR-based reporter construct to a concentration series of each saponin scored no EcR activation, while (2) a loss of ecdysteroid signaling was observed with median inhibitory concentrations (IC(50)'s) of 3-50 μM, and in parallel (3) a concentration-dependent change in loss of cell numbers in an cell viability assay with median effective concentrations (EC(50)'s) of 8-699 μM. In continuation, it was of interest that (4) a trypan blue assay with Q. saponaria saponin confirmed the cell membrane permeation effect leading to cell toxicity with a median lethal concentration (LC(50)) value of 44 μM, and interestingly this effect was very rapid. Another three interesting observations were that (5) exposure to 20E at 500 nM as used in the EcR-based report assay induced caspase-3 like activities which may help to explain the discrepancies between loss of EcR-responsiveness and cell viability, (6) low concentrations of saponins induced DNA fragmentation and caspase-3 like activities, confirming their potential to induce apoptosis, and (7) the saponin effects were counteracted with addition of cholesterol to the culture medium. In general the data obtained provide evidence that the anti-ecdysteroid action by saponins is not based on a true antagonistic interaction with EcR signaling, but can be explained by a cytotoxic action due to permeation of the

  16. Apollo 12 lunar material - Effects on lipid levels of tobacco tissue cultures.

    NASA Technical Reports Server (NTRS)

    Weete, J. D.; Walkinshaw, C. H.; Laseter, J. L.

    1972-01-01

    Tobacco tissue cultures grown in contact with lunar material from Apollo 12, for a 12-week period, resulted in fluctuations of both the relative and absolute concentrations of endogenous sterols and fatty acids. The experimental tissues contained higher concentrations of sterols than the controls did. The ratio of campesterol to stigmasterol was greater than 1 in control tissues, but less than 1 in the experimental tissues after 3 weeks. High relative concentrations (17.1 to 22.2 per cent) of an unidentified compound or compounds were found only in control tissues that were 3 to 9 weeks of age.

  17. Stimulating the production of homoisoflavonoids in cell suspension cultures of Caesalpinia pulcherrima using cork tissue.

    PubMed

    Zhao, Ping; Iwamoto, Yuko; Kouno, Isao; Egami, Yasukuni; Yamamoto, Hirobumi

    2004-09-01

    It has previously been demonstrated that cork tissue increases the efficiency of the production of lipophilic secondary metabolites in diverse plant cell suspension cultures. In the present study, three new homoisoflavonoids--named dihydrobonducellin, 2'-methoxydihydrobonducellin, and 2'-methoxybonducellin--and bonducellin and isobonducellin were isolated from Caesalpinia pulcherrima cultured cells coincubated with cork tissue. Cork tissue increased the production of 2'-methoxybonducellin by about 7-fold relative to control cells, and more than 80% of the product was recoverable from the cork tissue. When cork tissue and methyl jasmonate or yeast extract were added simultaneously to the medium, the amount of 2'-methoxybonducellin produced increased further. The production of the other four homoisoflavonoids was enhanced by variable amounts. Our results indicate that the addition of cork tissue would be an effective technique for investigating formation of secondary metabolites that usually accumulate only in trace amounts. PMID:15381409

  18. Expression of Ethylene Biosynthesis Genes in Barley Tissue Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant hormone ethylene influences green plant regeneration rates from barley callus cultures. Our studies have focused on the effects of short treatments of an ethylene inhibitor or an ethylene precursor on green plant regeneration from two barley cultivars and the expression patterns of two eth...

  19. Tissue Factor Activity in Lymphocyte Cultures from Normal Individuals and Patients with Hemophilia A

    PubMed Central

    Rickles, Frederick R.; Hardin, John A.; Pitlick, Frances A.; Hoyer, Leon W.; Conrad, Marcel E.

    1973-01-01

    The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant. PMID:4634046

  20. Polyphosphoinositides are present in plant tissue culture cells

    SciTech Connect

    Boss, W.F.; Massel, M.O.

    1985-11-15

    Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-(2-/sup 3/H) inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.

  1. Tissue culture system for infection with human hepatitis delta virus.

    PubMed Central

    Sureau, C; Jacob, J R; Eichberg, J W; Lanford, R E

    1991-01-01

    An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV. Images PMID:2041075

  2. The appropriateness of swab cultures for the release of human allograft tissue.

    PubMed

    Ronholdt, Chad J; Bogdansky, Simon

    2005-08-01

    Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference ( proportional, variant=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task. PMID:15973533

  3. Renal epithelia in long term gradient culture for biomaterial testing and tissue engineering.

    PubMed

    Minuth, Will W; Schumacher, Karl; Strehl, Raimund

    2005-01-01

    In the organism epithelia perform perfect barrier functions. Strong rheological and mechanical influences constitute the normal environment of this tissue throughout life. Most epithelia are exposed to different fluids at the luminal and basal sides. To obtain realistic information about tissue development in modern biomaterial testing and tissue engineering it is necessary to mimick the natural environment of epithelia. Cultured cells are brought in contact with an artificial extracellular matrix to determine whether proper development into a functional epithelium occurs. As under natural conditions the cultures have to withstand mechanical and fluid stress over a prolonged period of time in close contact to a selected biomaterial. However, development of tissue-specific features such as polarization, tightness and transport under in vitro conditions will only occur, if the biomaterial and the culture conditions support tissue development. Leakage, edge damage and pressure differences during culture have to be avoided so that the natural functions of the growing epithelium can develop. Our aim is to generate functional epithelia derived from renal explants containing stem cells, which are microsurgically isolated and placed into specific O-ring carriers for optimal handling. The cells develop in combination with a collagenous matrix from an embryonic into a functional collecting duct (rCD) epithelium. To achieve optimal culture conditions the tissue is placed in a gradient culture container. A typical environment can be simulated by superfusing different culture media at the luminal and basal sides. Within days epithelia growing inside the gradient container build up a physiological barrier, which is maintained during the whole culture period. The described method allows to investigate the influence of new biomaterials over prolonged periods of time. PMID:15623930

  4. Single molecule microscopy in 3D cell cultures and tissues.

    PubMed

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

    2014-12-15

    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. PMID:25453259

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

    PubMed Central

    Low, Eng-Ti L; Alias, Halimah; Boon, Soo-Heong; Shariff, Elyana M; Tan, Chi-Yee A; Ooi, Leslie CL; Cheah, Suan-Choo; Raha, Abdul-Rahim; Wan, Kiew-Lian; Singh, Rajinder

    2008-01-01

    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, in order to confirm

  6. Assessment of three types of spaceflight hardware for tissue culture studies: Comparison of skeletal tissue growth and differentiation

    NASA Astrophysics Data System (ADS)

    Klement, Brenda J.; Spooner, Brian S.

    1997-01-01

    Three different types of spaceflight hardware, the BioProcessing Module (BPM), the Materials Dispersion Apparatus (MDA), and the Fluid Processing Apparatus (FPA), were assessed for their ability to support pre-metatarsal growth and differentiation in experiments conducted on five space shuttle flights. BPM-cultured pre-metatarsal tissue showed no difference in flight and ground control lengths. Flight and ground controls cultured in the MDA grew 135 μm and 141 μm, respectively, in an 11 day experiment. Only five control rods and three flight rods mineralized. In another MDA experiment, pre-metatarsals were cultured at 4 °C (277K) or 20 °C (293K) for the 16 day mission, then cultured an additional 16 days in laboratory dishes at 37 °C (310K). The 20 °C (293K) cultures died post-flight. The 4 °C (277K) flight pre-metatarsals grew 417 μm more than the 4 °C (277K) ground controls post-flight. In 5 and 6 day experiments done in FPAs, flight rods grew longer than ground control rods. In a 14 day experiment, ground control and flight rods also expanded in length, but there was no difference between them. The pre-metatarsals cultured in the FPAs did not mineralize, or terminally differentiate. These experiments demonstrate, that while supporting pre-metatarsal growth in length, the three types of hardware are not suitable to support routine differentiation.

  7. Determinants of microstructural load transfer in cartilage tissue from chondrocyte culture

    NASA Astrophysics Data System (ADS)

    Fedewa, Michelle Marie

    2000-10-01

    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

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

    PubMed

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

    2016-01-01

    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. PMID:27123004

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

    PubMed Central

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

    2016-01-01

    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. PMID:27123004

  10. A Troubled Past? Reassessing Ethics in the History of Tissue Culture.

    PubMed

    Wilson, Duncan

    2016-09-01

    Recent books, articles and plays about the 'immortal' HeLa cell line have prompted renewed interest in the history of tissue culture methods that were first employed in 1907 and became common experimental tools during the twentieth century. Many of these sources claim tissue cultures like HeLa had a "troubled past" because medical researchers did not seek informed consent before using tissues in research, contravening a long held desire for self-determination on the part of patients and the public. In this article, I argue these claims are unfair and misleading. No professional guidelines required informed consent for tissue culture during the early and mid twentieth century, and popular sources expressed no concern at the widespread use of human tissues in research. When calls for informed consent did emerge in the 1970s and 1980s, moreover, they reflected specific political changes and often emanated from medical researchers themselves. I conclude by arguing that more balanced histories of tissue culture can make a decisive contribution to public debates today: by refuting a false dichotomy between science and its publics, and showing how ethical concepts such as informed consent arise from a historically specific engagement between professional and social groups. PMID:26240021

  11. Tissue culture and transient transformation of Marestail (Conyza canadensis (L.) Cronquist).

    PubMed

    Scheiber, P A; Tran, M; Duncan, D R

    2006-06-01

    Glyphosate resistant crops are useful to agriculture by facilitating the use of nonselective herbicides, such as RoundUp, that have low human and environmental toxicity. The occurrence of glyphosate resistant weeds, however, has raised concern about the future utility of these crops. Conyza canadensis (L.) Cronquist (marestail or horseweed) is one such glyphosate resistant weed that has yet to be fully analyzed or established in tissue culture. Tissue culture enables the examination of physiological characteristics of a plant in an aseptic and controlled environment. For the present study, mairstail was cultured on a Murashige and Skoog based medium supplemented with 6-benzylaminopurine, and alpha-naphthaleneacetic acid. Plant regeneration was achieved on the same basal medium supplemented with only gibberellic acid. Glyphosate resistance could be demonstrated in the cultured tissues. The cultures could also be transformed with Agrobacterium tumefaciens without chemically inducing virulence using phenolics or glucose and resulting in a transformation frequency (transgenic events per total number of explants used) of about 13%. The tissue culture growth, preliminary glyphosate resistance data and genetic transformation data gathered in this project provide the means to further evaluate the mode of glyphosate resistance expressed by marestail. PMID:16418861

  12. Trophoblast viability in perfused term placental tissue and explant cultures limited to 7-24 hours.

    PubMed

    Di Santo, S; Malek, A; Sager, R; Andres, A-C; Schneider, H

    2003-01-01

    Human term-placental culture techniques such as villous explant or dual perfusion are commonly used to study trophoblast function under control and experimentally manipulated conditions. We have compared trophoblast viability during perfusion and in explants cultured under various conditions by monitoring glucose consumption, protein synthesis and secretion, expression of differentiation-specific genes, induction of stress proteins and apoptotic cell death. The tissue was obtained from term-placentae of uncomplicated pregnancies after elective Caesarean delivery. We observed a severe loss of trophoblast viability in explants irrespective of the culture conditions used. Over 7 h of culture the amount of the differentiation specific placental hormones hCG, hPL and leptin accumulated in the medium dropped significantly. Analysis of their expression by semi-quantitative and real-time RT-PCR revealed that the down-regulation of expression occurred at the transcriptional level. This transcriptional repression was accompanied by induction of the stress-proteins RTP and BiP/GRP78. Analysis of apoptotic cell death by TUNEL assay and immunohistochemical detection of the caspase-3-specific degradation product of cytokeratin 18 revealed prominent cell death after 7 h of culture. These results are in contrast to the findings obtained in perfused placental tissue where, after 7 h of culture, hormone secretion, expression of stress proteins and cell death were similar as in native tissue. This difference between villous explant incubation and dual perfusion is also reflected by a significantly higher consumption of glucose in perfused tissue. PMID:13129686

  13. Imaging the division process in living tissue culture cells

    PubMed Central

    Khodjakov, Alexey; Rieder, Conly L.

    2008-01-01

    We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy. PMID:16343936

  14. Inheritance and expression of lysine plus threonine resistance selected in maize tissue culture

    PubMed Central

    Hibberd, K. A.; Green, C. E.

    1982-01-01

    Selection for resistance to growth inhibition by lysine plus threonine in equimolar concentrations (LT) in tissue cultures of maize yielded a stable resistant line, LT19. Genetic analysis of progeny of plants regenerated from LT19 showed that LT resistance was inherited as a single dominant nuclear gene, temporarily designated Ltr*-19. Tissue cultures initiated from resistant embryos required 5-10 times higher levels of LT to inhibit growth than did cultures from LT-sensitive embryos. LT resistance in Ltr*-19 was expressed as much reduced inhibition of root and shoot growth in the presence of LT. The free pool of threonine was increased 6 times in cultures initiated from immature embryos of LT-resistant plants, and 75-100 times in kernels homozygous for Ltr*-19, as compared to cultures and kernels from LT-sensitive embryos and plants, respectively. Overproduction of free threonine increased the total threonine content in homozygous Ltr*-19 kernels by 33-59%. The results demonstrate that LT resistance selected with tissue culture methods is heritable and is expressed in cultures, seedlings, and kernels. Furthermore, they demonstrate a method to obtain amino acid-overproducer mutants in maize, which have the potential to increase substantially specific amino acids in kernels. The capability to increase specifically the nutritionally limiting amino acid(s) could have important nutritional implications for the grain of cereals and other crops. PMID:16593148

  15. Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

    PubMed Central

    Storm, Michael P.; Sorrell, Ian; Shipley, Rebecca; Regan, Sophie; Luetchford, Kim A.; Sathish, Jean; Webb, Steven; Ellis, Marianne J.

    2016-01-01

    Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture. PMID:27285826

  16. Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture.

    PubMed

    Storm, Michael P; Sorrell, Ian; Shipley, Rebecca; Regan, Sophie; Luetchford, Kim A; Sathish, Jean; Webb, Steven; Ellis, Marianne J

    2016-01-01

    Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture. PMID:27285826

  17. Tissue culture system using a PANDA ring resonator and wavelength router for hydroponic plant.

    PubMed

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

    2012-06-01

    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. PMID:22411055

  18. Radiosensitivity of different tissues from carrot root at different phases of growth in culture

    SciTech Connect

    Degani, N.; Pickholtz, D.

    1980-09-01

    The present work compares the effect of ..gamma..-radiation dose and time in culture on the growth of cambium and phloem carrot (Daucus carota) root explants. It was found that the phloem is more radiosensitive than the cambium and that both tissues were more radiosensitive when irradiated on excision at the G/sub 1/ phase rather than at the end of the lag phase on the ninth day of growth in culture when cells were predominantly at the G/sub 2/ phase. The nuclear volumes of cells from both tissues were similar but were larger at the end of the more radioresistant lag phase than those of the G/sub 1/ phase on excision. However, nuclear volume could not account for the differences in radiosensitivity between either the tissues or irradiation times in culture.

  19. Quantitation of ranaviruses in cell culture and tissue samples.

    PubMed

    Holopainen, Riikka; Honkanen, Jarno; Jensen, Britt Bang; Ariel, Ellen; Tapiovaara, Hannele

    2011-01-01

    A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV. PMID:21087639

  20. Clinical and morphological investigations on ependymomas and their tissue cultures.

    PubMed

    Casentini, L; Gullotta, F; Möhrer, U

    1981-03-01

    A morphological investigation was carried out on 56 ependymomas cultivated in vitro as short-term cultures in roller tubes. The tumours had been histologically classified as cellular and fibrillary ependymomas, subependymomas, myxopapillary and malignant ependymomas (Table 1). A very good growth was detected in 32 cases, most of them being cellular and malignant ependymomas (Table 2). The prevailing growth pattern was epithelial in type, i.e. proliferating cells forming a carpet. In some cases, in the first stages of growth elongated bipolar cells did appear, but they evolved later as flattened epithelial elements. In four cases, a mixed proliferation of piloid astrocytes and ependymal cells was seen; these tumours were regarded as mixed gliomas. In 46 cases an exact evaluation of the history was possible. Although no correlation could be found between histology and survival time (Table 4), the longest survival was observed in spinal tumours (Table 3). Tumours in children had a slightly worse prognosis in comparison with adults (Table 5). A radical removal of the tumour was generally followed by a longer survival time (Table 6), although the operative procedure employed did not seem to influence the development of recurrences (Table 7). PMID:7219656

  1. Extracts from black carrot tissue culture as potent anticancer agents.

    PubMed

    Sevimli-Gur, Canan; Cetin, Burcu; Akay, Seref; Gulce-Iz, Sultan; Yesil-Celiktas, Ozlem

    2013-09-01

    Black carrots contain anthocyanins possessing enhanced physiological activities. Explants of young black carrot shoots were cultured in Murashige and Skoog (MS) medium for callus initiation and were transferred to new MS medium supplemented with four different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Subsequently, the lyophilized calli and black carrot harvested from fields were subjected to ultrasound extraction with ethanol at a ratio of 1:15 (w:v). Obtained extracts were applied to various human cancer cell lines including MCF-7 SK-BR-3 and MDA-MB-231 (human breast adenocarcinomas), HT-29 (human colon adenocarcinoma), PC-3 (human prostate adenocarcinoma), Neuro 2A (Musmusculus neuroblastoma) cancer cell lines and VERO (African green monkey kidney) normal cell line by MTT assay. The highest cytotoxic activity was achieved against Neuro-2A cell lines exhibiting viability of 38-46% at 6.25 μg/ml concentration for all calli and natural extracts. However, a significantly high IC50 value of 170.13 μg/ml was attained in normal cell line VERO indicating that its natural counterpart is an ideal candidate for treatment of brain cancer without causing negative effects to normal healthy cells. PMID:23828497

  2. A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

    PubMed Central

    Parikh, Mukti R.; Belch, Andrew R.; Pilarski, Linda M; Kirshner, Julia

    2014-01-01

    Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions

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

    SciTech Connect

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M. )

    1990-04-01

    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.

  4. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    PubMed Central

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  5. Studies on Japanese B Encephalitis Virus Vaccines from Tissue Culture

    PubMed Central

    Singh, Balwant; Hammon, W. McD.

    1971-01-01

    A study was carried out to evaluate the reliability of and to determine the mechanism involved in an antigen extinction mouse intraperitoneal (ip) challenge test for potency of a cell culture vaccine for Japanese B encephalitis, a modification of a test originated by Sabin for a mouse brain vaccine. Some comparisons were made with the official Japanese test using an intracerebral (ic) challenge after a more prolonged immunization procedure. The Japanese method of using a lyophilized reference vaccine with each test was also employed. It was found that the ip and the ic test appeared to show similar relative differences between lots. The ip test was more quickly and readily performed, gave reasonably consistent results on repetition, and, when used with a suitable reference vaccine, gave promise of being an entirely suitable and reliable test. Immunization by the intramuscular route rather than by the regular ip route appeared to offer no advantage and was less consistent in responses shown. Neutralizing antibody responses of the mice in the standard procedure were very quick to appear, about 4 days after the first dose of vaccine and had a peak titer about the seventh day, the time of challenge. This titer fell quickly unless challenge occurred. The antibody was heat stable, but it was readily inactivated by 2-mercaptoethanol (2-ME). Not until the 11th or 15th day did a small amount of immunoglobulin G appear. Challenge on day 7 significantly increased titers, but this antibody was also mostly inactivated by 2-ME. Interferon did not appear to play any significant role in the protection shown by the mice. PMID:4325023

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

    SciTech Connect

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. )

    1990-10-01

    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.

  7. Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study

    PubMed Central

    Bernik, Maria B.; Kwaan, Hau C.

    1969-01-01

    Human tissues and cells from pre- and postnatal life were cultivated and studied for plasminogen activator activity. Cultures were obtained from kidney, renal blood vessels, ureter, bladder, lung, and heart. Local activator activity of cells was demonstrated by histochemical techniques. Activator released by cells into the supernatant culture media was assayed by fibrin plate techniques and was investigated for immunological identity using specific antisera to an activator of human origin, urokinase (UK). Plasminogen activator was produced in primary cultures where cells retain specific functions and generally reflect the enzyme pattern of the tissues of origin. Cells from fetal and adult sources were found to yield activator antigenically identical to UK, as well as activator activity which differed from that of UK in immunoassays and which may represent tissue type activator. Such activity was released after injury or death of cells while UK was produced in cultures containing live, metabolizing cells. Primary cultures of kidney confirmed that this organ is a rich source of UK and demonstrated, in addition, that UK is produced from the early stages of gestation and in increasing amounts thereafter. However, primary cultures also demonstrated that the ability to produce UK is not limited to the kidney but is a function of cells which are distributed widely in body tissues. Thus, activator antigenically identical to UK accumulated progressively after many refeedings in culture supernates of fetal lung and ureter, as well as in supernates of renal blood vessels of adults. These findings indicate continuous formation of UK by the cultured cells and, furthermore, provide evidence of UK production in blood vessels. In cultures from other tissues, particularly those from fetal heart and adult lung and bladder, investigation of activator was hindered by inhibitory activity which accumulated in the supernates. Such activity was derived from cells in culture and was

  8. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

    PubMed

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-Ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

    2014-09-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. PMID:25063093

  9. Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

    PubMed Central

    2010-01-01

    Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral

  10. Vascularized Bone Tissue Formation Induced by Fiber-Reinforced Scaffolds Cultured with Osteoblasts and Endothelial Cells

    PubMed Central

    Liu, Xinhui; Zhang, Guoping; Hou, Chuanyong; Wang, Hua; Yang, Yelin; Guan, Guoping; Dong, Wei; Gao, Hongyang

    2013-01-01

    The repair of the damaged bone tissue caused by damage or bone disease was still a problem. Current strategies including the use of autografts and allografts have the disadvantages, namely, diseases transmission, tissue availability and donor morbidity. Bone tissue engineering has been developed and regarded as a new way of regenerating bone tissues to repair or substitute damaged or diseased ones. The main limitation in engineering in vitro tissues is the lack of a sufficient blood vessel system, the vascularization. In this paper, a new-typed hydroxyapatite/collagen composite scaffold which was reinforced by chitosan fibers and cultured with osteoblasts and endothelial cells was fabricated. General observation, histological observation, detection of the degree of vascularization, and X-ray examination had been done to learn the effect of vascularized bone repair materials on the regeneration of bone. The results show that new vessel and bone formed using implant cultured with osteoblasts and endothelial cells. Nanofiber-reinforced scaffold cultured with osteoblasts and endothelial cells can induce vascularized bone tissue formation. PMID:24369019

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

    NASA Astrophysics Data System (ADS)

    Arlinghaus, H. F.; Kriegeskotte, C.; Fartmann, M.; Wittig, A.; Sauerwein, W.; Lipinsky, D.

    2006-07-01

    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 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.

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

    PubMed Central

    Röder, Alexander; García-Gareta, Elena; Theodoropoulos, Christina; Ristovski, Nikola; Blackwood, Keith A.; Woodruff, Maria A.

    2015-01-01

    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. PMID:26703748

  13. Tissue-culture investigations into mechanisms of biomass enhancement. Annual report, June 1984-July 1985

    SciTech Connect

    Nabors, M.W.

    1985-07-01

    The cost effectiveness of biogas production can be considerably improved by producing cultivars of sorghum and Napier grass with increased biomass and tolerance to common soil stresses such as salinity and drought. In addition, increased fertilizer efficiency of plants used for biomass is also desired. Tissue-culture methodologies provide a means for generating improved sorghum and Napier grass cultivars and for selecting cells and plants with tolerance to salinity, drought, and low levels of applied nitrogen fertilizer. To this end, tissue cultures of sorghum and Napier grass were established. Media were devised to enhance high-frequency, long-term plant production from these cultures. Existing methods were considerably improved and the first plant regeneration techniques from callus cultures of sweet sorghum were devised. Over 1000 plants were regenerated from callus cultures during the first year. These are being used in biomass production assays. Tissue culture selection for salt tolerance has been initiated using high levels of NaCl or hydroxyproline in the medium. Sodium chloride stress represents direct selection; hydroxyproline stress selects cells with increased levels of proline, an amino acid known to be associated with salt tolerance. Selection for cell variants efficient in reducing nitrate are planned; cells will be grown in the presence of chlorate, a nitrate analogue. Selections are carried out on either solid or liquid media. Cell suspension systems, allowing more efficient selection, are being developed for all cultivars under study.

  14. The Porcine Aortic Tissue Culture System in vitro for Stem Cell Research

    PubMed Central

    Kim, Dong-Eun; Oh, Keun-Hee; Yang, Ji-Hye; Kwon, Sun-Keun; Cho, Tae-Jun; Lee, Seul-Bi; Nam, Hyun; Lee, Dong-Sup; Lee, Jung-Ryul; Lee, Gene

    2011-01-01

    Background and Objectives: Due to the shortage of human donors for transplantation, the use of animal organs for xenotransplantation has come into great interest. Xeno-derived vessels and cardiac valves would be possible alternatives for the patient suffering from cardiovascular diseases. Therefore, we established in vitro culture system of a porcine vessel that could be helpful for the research of xenograft and stem cell research. Methods and Results: We primarily isolated porcine thoracic aorta, cultured square-shaped pieces up to 17 days and analyzed its morphology and characters. The endothelial cells were primarily isolated from cultured porcine aortic pieces and their morphology, function and character were analyzed in order to confirm them as endothelial cells at day 3, 4, 8, 10 and 17. Even at day 17, the morphology exhibited the intact endothelial layer as well as specifically expressed CD31 and von Willebrand factor. The morphology of primarily isolated cells from cultured tissues was identical as an endothelial cell. By flow cytometry analysis, more than 80% of the isolated cells expressed CD31 and up to 80% took up acetyl low density lipoprotein (ac-LDL) until day 10 of tissue culture period even though it decreased to about 50% at day 17 that means they not only showed typical endothelial cell characters but also functioned properly. Conclusions: We successfully established and optimized a porcine vascular tissue in vitro culture system that could be a valuable model for in vitro study of xenotransplantation and stem cell research. PMID:24298344

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

    PubMed

    Röder, Alexander; García-Gareta, Elena; Theodoropoulos, Christina; Ristovski, Nikola; Blackwood, Keith A; Woodruff, Maria A

    2015-01-01

    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. PMID:26703748

  16. Explant culture of gastrointestinal tissue: a review of methods and applications.

    PubMed

    Randall, Kevin J; Turton, John; Foster, John R

    2011-08-01

    The gastrointestinal (GI) tract is an important target organ for the toxicity of xenobiotics. The toxic effects of xenobiotics on this complex, heterogeneous structure have been difficult to model in vitro and have traditionally been assessed in vivo. The explant culture of GI tissue offers an alternative approach. Historically, the organotypic culture of the GI tract proved far more challenging than the culture of other tissues, and it was not until the late 1960s that Browning and Trier described the means by which intestinal tissues could be successfully cultured. This breakthrough provided a tool researchers could utilise, and adapt, to investigate topics such as the pathogenesis of inflammatory intestinal diseases, the effect of growth factors and cytokines on intestinal proliferation and differentiation, and the testing of novel xenobiotics for efficacy and safety. This review considers that intestinal explant culture shows much potential for the application of a relatively under-used procedure in future biomedical research. Furthermore, there appear to be many instances where the technique may provide experimental solutions where both cell culture and in vivo models have been unable to deliver conclusive and convincing findings. PMID:21384137

  17. Production of Limonoids with Insect Antifeedant Activity in a Two-Stage Bioreactor Process with Cell Suspension Culture of Azadirachta indica.

    PubMed

    Vásquez-Rivera, Andrés; Chicaiza-Finley, Diego; Hoyos, Rodrigo A; Orozco-Sánchez, Fernando

    2015-09-01

    Neem tree (Azadirachta indica) cell suspension culture is an alternative for the production of limonoids for insect control that overcomes limitations related to the supply of neem seeds. To establish conditions for cell growth and azadiracthin-related limonoid production, the effect of different sucrose concentrations, nitrate and phosphate in Murashige and Skoog (MS) medium, and the addition of one precursor and three elicitors was evaluated in shake flasks. The process was scaled up to a 3-l stirred tank bioreactor in one- and two-stage batch cultivation. In shake flasks, more than fivefold increase in the production of limonoids with the modified MS medium was observed (increase from 0.77 to 4.52 mg limonoids/g dry cell weight, DCW), while an increase of more than fourfold was achieved by adding the elicitors chitosan, salicylic acid, and jasmonic acid together (increase from 1.03 to 4.32 mg limonoids/g DCW). In the bioreactor, the volumetric production of limonoids was increased more than threefold with a two-stage culture in day 18 (13.82 mg limonoids/l in control single-stage process and 41.44 mg/l in two-stage process). The cultivation and operating mode of the bioreactor reported in this study may be adapted and used in optimization and process plant development for production of insect antifeedant limonoids with A. indica cell suspension cultures. PMID:26234433

  18. Mycetocyte symbiosis in insects.

    PubMed

    Douglas, A E

    1989-11-01

    1. Non-pathogenic microorganisms, known as mycetocyte symbionts, are located in specialized 'mycetocyte' cells of many insects that feed on nutritionally unbalanced or poor diets. The insects include cockroaches, Cimicidae and Lygaeidae (Heteroptera), the Homoptera, Anoplura, the Diptera Pupiparia, some formicine ants and many beetles. 2. Most mycetocyte symbionts are prokaryotes and a great diversity of forms has been described. None has been cultured in vitro and their taxonomic position is obscure. Yeasts have been reported in Cerambycidae and Anobiidae (Coleoptera) and a few planthoppers. They are culturable and those in anobiids have been assigned to the genus Torulopsis. 3. The mycetocyte cells may be associated with the gut, lie free in the abdominal haemocoel or be embedded in the fat body of the insect. The mycetocytes are large polyploid cells which rarely divide and the symbionts are restricted to their cytoplasm. 4. The mycetocyte symbionts are transmitted maternally from one insect generation to the next. In many beetles (Anobiidae, Cerambycidae, Chrysomelidae and cleonine Curculionidae), the microoganisms are smeared onto the eggs and consumed by the hatching larvae. In other insects, they are transferred from mycetocytes to oocytes in the ovary, a process known as transovarial transmission. The details of transmission in the different insect groups vary with the age of the mother (adult, larva or embryo) at which symbiont transfer to the ovary is initiated; whether isolated symbionts or intact mycetocytes are transferred; and the site of entry of symbionts to the egg (anterior, posterior or apolar). 5. Within an individual insect, the biomass of symbionts varies in a regular fashion with age, weight and sex of the insect. Suppression of symbiont growth rate and lysis of 'excess' microorganisms may contribute to the regulation of symbionts (including freshly-isolated preparations of unculturable forms) are used to investigate interactions between the

  19. Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

    PubMed Central

    Bhagavathula, Narasimharao; Mankey, Cohra; DaSilva, Marissa; Paruchuri, Tejaswi; Aslam, Muhammad Nadeem; Varani, James

    2009-01-01

    Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO2 and 95% O2; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca2+ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue. PMID:19915935

  20. Participation of cob tissue in the transport of medium components into maize kernels cultured in vitro

    SciTech Connect

    Felker, F.C. )

    1990-05-01

    Maize (Zea mays L.) kernels cultured in vitro while still attached to cob pieces have been used as a model system to study the physiology of kernel development. In this study, the role of the cob tissue in uptake of medium components into kernels was examined. Cob tissue was essential for in vitro kernel growth, and better growth occurred with larger cob/kernel ratios. A symplastically transported fluorescent dye readily permeated the endosperm when supplied in the medium, while an apoplastic dye did not. Slicing the cob tissue to disrupt vascular connections, but not apoplastic continuity, greatly reduced ({sup 14}C)sucrose uptake into kernels. ({sup 14}C)Sucrose uptake by cob and kernel tissue was reduced 31% and 68%, respectively, by 5 mM PCMBS. L-({sup 14}C)glucose was absorbed much more slowly than D-({sup 14}C)glucose. These and other results indicate that phloem loading of sugars occurs in the cob tissue. Passage of medium components through the symplast cob tissue may be a prerequisite for uptake into the kernel. Simple diffusion from the medium to the kernels is unlikely. Therefore, the ability of substances to be transported into cob tissue cells should be considered in formulating culture medium.

  1. Assessment of three types of spaceflight hardware for tissue culture studies: Comparison of skeletal tissue growth and differentiation

    SciTech Connect

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

    1997-01-01

    Three different types of spaceflight hardware, the BioProcessing Module (BPM), the Materials Dispersion Apparatus (MDA), and the Fluid Processing Apparatus (FPA), were assessed for their ability to support pre-metatarsal growth and differentiation in experiments conducted on five space shuttle flights. BPM-cultured pre-metatarsal tissue showed no difference in flight and ground control lengths. Flight and ground controls cultured in the MDA grew 135 {mu}m and 141 {mu}m, respectively, in an 11 day experiment. Only five control rods and three flight rods mineralized. In another MDA experiment, pre-metatarsals were cultured at 4{degree}C (277K) or 20{degree}C (293K) for the 16 day mission, then cultured an additional 16 days in laboratory dishes at 37{degree}C (310K). The 20{degree}C (293K) cultures died post-flight. The 4{degree}C (277K) flight pre-metatarsals grew 417 {mu}m more than the 4{degree}C (277K) ground controls post-flight. In 5 and 6 day experiments done in FPAs, flight rods grew longer than ground control rods. In a 14 day experiment, ground control and flight rods also expanded in length, but there was no difference between them. The pre-metatarsals cultured in the FPAs did not mineralize, or terminally differentiate. These experiments demonstrate, that while supporting pre-metatarsal growth in length, the three types of hardware are not suitable to support routine differentiation. {copyright} {ital 1997 American Institute of Physics.}

  2. The in vitro immunoregulatory properties of cultured murine trophoblast are not unique to this tissue.

    PubMed Central

    Drake, B L; Rodger, J C

    1985-01-01

    Primary cultures of murine trophoblast (ectoplacental cone and mid-term placenta) and their supernatants were found to inhibit in vitro lymphocyte proliferative responses to concanavalin A (77-87%) and allo-antigen (52-84%). However, cultures and cell-conditioned media from non-trophoblastic tissues (embryonic sac, adult lung and liver, and B16 melanoma line) produced similar results. In all cases, the inhibitory effects were not due to reduced cell viability. Addition of anti-progesterone serum to the ectoplacental cone-lymphocyte co-cultures, at a concentration known to bind the available trophoblast-derived progesterone, did not overcome the observed suppression. The results clearly demonstrate that a range of cultured cell types, and their conditioned media, will suppress immune responses in vitro. We conclude that cultured trophoblast is not an appropriate model for studies of placental immunoregulation. PMID:3159651

  3. Tissue-Specific Cultured Human Pericytes: Perivascular Cells from Smooth Muscle Tissue Have Restricted Mesodermal Differentiation Ability.

    PubMed

    Pierantozzi, Enrico; Vezzani, Bianca; Badin, Margherita; Curina, Carlo; Severi, Filiberto Maria; Petraglia, Felice; Randazzo, Davide; Rossi, Daniela; Sorrentino, Vincenzo

    2016-05-01

    Microvascular pericytes (PCs) are considered the adult counterpart of the embryonic mesoangioblasts, which represent a multipotent cell population that resides in the dorsal aorta of the developing embryo. Although PCs have been isolated from several adult organs and tissues, it is still controversial whether PCs from different tissues exhibit distinct differentiation potentials. To address this point, we investigated the differentiation potentials of isogenic human cultured PCs isolated from skeletal (sk-hPCs) and smooth muscle tissues (sm-hPCs). We found that both sk-hPCs and sm-hPCs expressed known pericytic markers and did not express endothelial, hematopoietic, and myogenic markers. Both sk-hPCs and sm-hPCs were able to differentiate into smooth muscle cells. In contrast, sk-hPCs, but not sm-hPCs, differentiated in skeletal muscle cells and osteocytes. Given the reported ability of the Notch pathway to regulate skeletal muscle and osteogenic differentiation, sk-hPCs and sm-hPCs were treated with N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a known inhibitor of Notch signaling. DAPT treatment, as assessed by histological and molecular analysis, enhanced myogenic differentiation and abolished osteogenic potential of sk-hPCs. In contrast, DAPT treatment did not affect either myogenic or osteogenic differentiation of sm-hPCs. In summary, these results indicate that, despite being isolated from the same anatomical niche, cultured PCs from skeletal muscle and smooth muscle tissues display distinct differentiation abilities. PMID:26956507

  4. Changes in delta 13C stable isotopes in multiple tissues of insect predators fed isotopically distinct prey.

    PubMed

    Gratton, Claudio; Forbes, Andrew E

    2006-04-01

    Traditionally, researchers have used measurements of carbon stable isotopes to infer the composition of consumers' diets. However, since consumer's tissues may process carbon isotopes differently, particularly following a diet shift, it is possible to use measurements of carbon isotopes in multiple tissues to determine not only the composition of an individual's diet, but also the temporal dynamics thereof. This study examined how stable isotopes of carbon (13C/12C, expressed as delta 13C) changed in different adult tissues of two predacious beetles, Harmonia axyridis and Coccinella septempunctata (Coleoptera: Coccinellidae). In the laboratory, we switched ladybeetles from a C3-based diet (soybean aphids, Aphis glycines) to a C4-based one (corn leaf aphids, Rhopalosiphum maidis). The delta 13C of metabolically active tissues such as the body fat and reproductive organs changed rapidly (< or =5 days) following the diet shift. Tissues expected to be more metabolically inert, such as wings, changed more slowly over the same period. Although these general patterns were largely similar between males and females, females had more rapid changes in delta 13C in fat and reproductive tissues. However, females showed a significant depletion in delta 13C after 10 days, while males' delta 13C continued to increase. Given the results of this experiment, it is now possible to distinguish between ladybeetles eating a mixed diet (beetles with multiple tissues at similar, intermediate, equilibrial delta 13C signatures) from those that have shifted diets (beetles with different tissues at distinctly different delta 13C values). Thus, this approach can be used broadly to infer not only what constitutes the diet of a consumer, but also the temporal history of dietary intake. PMID:16341886

  5. Using Peat Pellets in Liquid Media to Root Sunflower Tissue Culture Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditional plant breeding is often limited by the genetic diversity within a species. The use of biotechnology allows introducing into a plant, specific traits that come from the same or another plant species. In this paper, we focus on tissue culture of sunflower (Helianthus annus L., Asteraceae...

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

    NASA Astrophysics Data System (ADS)

    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 < 0.05) different. Both irrigation regimes resulted in significant ( P < 0.05) increases in leaf number, leaf length, leaf area, leaf area index, green leaf colour intensity, 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.

  7. Extended metAFLP approach in studies of tissue culture induced variation (TCIV) in triticale.

    PubMed

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

    2014-01-01

    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. PMID:25242884

  8. Potato transformation and potato cyst nematode infection on potato plantlets in tissue culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    These two protocols describe the methods for generating transgenic potato plants and for evaluating potato cyst nematode (Globodera rostochiensis and G. pallida) infection on potato plantlets in tissue culture. These methods are useful tools that can be used in the study of the interactions between ...

  9. Expression Analysis of Ethylene Biosynthesis and Receptor Genes From Barley Embryo and Tissue Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethylene affects regeneration of green plants from barley tissue culture. With the availability of the HarvEST barley database and barley GeneChip, genome-wide expression studies have focused on differential development between Morex and Golden Promise at various stages of plant growth. The data f...

  10. Amending Storage Vessel and Media Improves Subculture Interval of Musa sp. Tissue Culture Plantlets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. In Situ Immune Infrared Fluorescent Staining for Detection and Quantification of Bluetongue Virus in Cullicoides Insect Cell Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from C. sonorensis; however, techniques for directly detecting and quantifying virus in these insect cells are lacking. In situ immune infrared fluorescent s...

  12. In situ immune infrared fluorescent staining for detection and quantification of bluetongue virus in insect cell culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines developed from C. sonorensis have facilitated studies of virus replication and the role of the insect vector in BTV transmission. However, techniques for directly detecting viru...

  13. Enterococci in Insects

    PubMed Central

    Martin, Jonathan D.; Mundt, J. Orvin

    1972-01-01

    Enterococci were obtained from 213 of 403 insects cultured during a 14-month period, in numbers from 103 to 3 × 107/g of insect. Insects were taken only from nonurban, wild, and cultivated fields and woods. In species of insects carrying them, enterococci were not always present in every individual cultured, and often more than one species of enterococcus occurred within a species. Enterococci were obtained from certain insects taken in the field during the dormant season, suggesting their role as overwintering agents. They were generally present in species feeding on nectar, succulent plant parts, and on and ir forest litter, but not from insects feeding on less succulent leaves and stems. Streptococcus faecalis was recovered from 32%, Streptococcus faecium from 22.4%, and Streptococcus faecium var. casseliflavus from 43.5% of members of the 37 taxa of insects. S. faecalis and S. faecium var. casseliflavus exhibit a high percent of conformity to the properties published for them. The heterogeneity in properties of S. faecium is similar to that found for the species taken from plants. Many fail to grow in broth at 45 C or in broth containing 6.5% NaCl; 50% of the cultures ferment both melezitose and melibiose, and a few ferment neither sugar. The remainder ferment melibiose only. Failure to reduce methylene blue in milk by S. faecalis and S. faecium is correlated with the inability to ferment lactose. More than 93% of the cultures of S. faecalis digest casein in milk from the top downward, following the production of a soft, flowing curd. Because this property is not characteristic of S. faecalis taken from humans, the reaction in litmus milk is suggested as a means of differentiation between cultures of remote and innocent origin in nature and recent, human pollution. PMID:4628796

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

    PubMed Central

    Bednarek, Piotr T; Orłowska, Renata; Koebner, Robert MD; Zimny, Janusz

    2007-01-01

    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 species which are sexually

  15. Tissue culture characteristics of maize (Zea mays L.) haploid coleoptile sections.

    PubMed

    Jiang, L; Jing, G X; Li, X Y; Wang, X Q; Xing, Z; Deng, P K; Zhao, R G

    2015-01-01

    Doubled haploid (DH) technology, which is used for rapidly purifying genetic resources, is a key technology in modern maize breeding. The present study evaluated the tissue culture characteristics of maize haploid coleoptile sections, in order to provide a new way of haploid doubling. With 20 combinations of haploid coleoptile sections, obtained by hybridization within Reid, Tangsipingtou, and Term-tropical groups, as explants, we analyzed the induction and differentiation rate of callus, observed the number of root tip chromosomes in regenerated plants, and analyzed the pollen fertility. In addition, we used 47 SSR markers to analyze the genotypes of regenerated plants. The Reid and Tangsipingtou groups had significantly higher induction rates of haploid coleoptile callus compared to the Term-tropical group. Fifteen haploid plants were obtained which had 10 chromosomes in the root tips as assessed by I-KI staining. It was also noticed that the pollen of pollinated anthers were partially fertile. The haploid plants had genetic stability and showed no variation. The Reid and Tangsipingtou groups had good culture characteristics of haploid coleoptile sections, while the Term-tropical group had poor culture characteristics. Genotypes of haploid plants generated by tissue culture were evidenced to come from recombinant types of parents. Thus, this study established a tissue culture system of maize haploid coleoptile. PMID:26662420

  16. Optimizing culture medium for meristem tissue culture of several Saccharum species and commercial hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  17. Design and validation of a biomechanical bioreactor for cartilage tissue culture.

    PubMed

    Correia, V; Panadero, J A; Ribeiro, C; Sencadas, V; Rocha, J G; Gomez Ribelles, J L; Lanceros-Méndez, S

    2016-04-01

    Specific tissues, such as cartilage, undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation--amplitude and frequency--to three-dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells. PMID:26153426

  18. An Alternative Gelling Agent for Culture and Studies of Nematodes, Bacteria, Fungi, and Plant Tissues

    PubMed Central

    Ko, M. P.; Van Gundy, S. D.

    1988-01-01

    Pluronic F127 polyol, a block copolymer of propylene oxide and ethylene oxide, was studied as an alternative to agar in culture media for nematodes, bacteria, fungi, actinomycetes, and plant tissues or seedlings, At a polyol concentration of 20% w/v, the culture media, semi-solid at room temperature (22 C) but liquid at lower temperatures, had minimal effects on the test organisms. Most of the fungi and bacteria grew as well in 20% polyol as in 1.5% agar media; however, various species of nematodes and plant seedlings or tissues exhibited differential sensitivities to different concentrations of the polyol. In cases where the organisms were unaffected, the polyol media had certain advantages over agar, including greater transparency and less contamination under nonaseptic conditions. Polyol media have potentially greater ease for recovery of embedded organisms or tissues inside the media by merely shifting to lower temperatures. PMID:19290241

  19. Distribution of phospholipase C isozymes in various rat tissues and cultured cells

    SciTech Connect

    Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

    1987-05-01

    Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts.

  20. [The three-dimensional culture of adult mesenchymal stem cells for intervertebral disc tissue engineering].

    PubMed

    Feng, Ganjun; Liu, Hao; Deng, Li; Chen, Xiaohe; Zhao, Xianfeng; Liang, Tao; Li, Xiuqiong

    2009-12-01

    Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. As current clinical treatments are aimed at restoring biomechanical function and providing symptomatic relief, the methods focused on biological repair have aroused interest and several tissue engineering approaches using different cell types have been proposed. Owing to the unsuitable nature of degenerate cells for tissue engineering, attention has been given to the use of mesenchymal stem cells (MSCs). In this connection, we have made a study on the characteristics of MSCs derived from adult bone marrow and on the feasibility of constructing IVD tissue-engineering cell under a Three-Dimensional Pellet Culture System. The human bone marrow MSCs were isolated and purified with density gradient solution and attachment-independent culture system. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria. They have the capacity for self-renewal and proliferation, and the multilineage potential to differentiate. PMID:20095491

  1. On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

    PubMed

    Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

    2016-07-01

    Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications. PMID:27493703

  2. Oxidative phosphorylation and mitochondrial function differ between human prostate tissue and cultured cells.

    PubMed

    Schöpf, Bernd; Schäfer, Georg; Weber, Anja; Talasz, Heribert; Eder, Iris E; Klocker, Helmut; Gnaiger, Erich

    2016-06-01

    Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism. PMID:27060259

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

    ERIC Educational Resources Information Center

    Peat, Gerry; Jones, Meriel

    2012-01-01

    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…

  4. Electrospun fibrinogen: feasibility as a tissue engineering scaffold in a rat cell culture model.

    PubMed

    McManus, Michael C; Boland, Eugene D; Simpson, David G; Barnes, Catherine P; Bowlin, Gary L

    2007-05-01

    Fibrinogen has a well-established tissue engineering track record because of its ability to induce improved cellular interaction and scaffold remodeling compared to synthetic scaffolds. While the feasibility of electrospinning fibrinogen scaffolds of submicron diameter fibers and their mechanical properties have been demonstrated, in vitro cellular interaction has not yet been evaluated. The goal of this study was to demonstrate, based on cellular interaction and scaffold remodeling, that electrospun fibrinogen can be used successfully as a tissue engineering scaffold. Electrospun fibrinogen scaffolds were disinfected, seeded with neonatal rat cardiac fibroblasts, and cultured for 2, 7, and 14 days. Cultures were treated to regulate scaffold degradation by either supplementing serum-containing media with aprotinin or crosslinking the scaffolds with glutaraldehyde vapor. Biocompatibility was assessed through a WST-1 cell proliferation assay. Postculture scaffolds were evaluated by scanning electron microscopy and histology. Cell culture demonstrated that fibroblasts readily migrate into and remodel electrospun fibrinogen scaffolds with deposition of native collagen. Supplementation of culture media with different concentrations of aprotinin-modulated scaffold degradation in a predictable fashion, but glutaraldehyde vapor fixation was less reliable. Based on the observed cellular interactions, there is tremendous potential for electrospun fibrinogen as a tissue engineering scaffold. PMID:17120217

  5. Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

    PubMed

    Nato, A; Mirshahi, A; Tichtinsky, G; Mirshahi, M; Faure, J P; Lavergne, D; De Buyser, J; Jean, C; Ducreux, G; Henry, Y

    1997-03-01

    An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed. PMID:9085574

  6. Organ and tissue donation in migrants: advanced course for cross-cultural mediators.

    PubMed

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

    2013-09-01

    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. PMID:24033996

  7. Immunological detection of potential signal-transduction proteins expressed during wheat somatic tissue culture.

    PubMed Central

    Nato, A; Mirshahi, A; Tichtinsky, G; Mirshahi, M; Faure, J P; Lavergne, D; De Buyser, J; Jean, C; Ducreux, G; Henry, Y

    1997-01-01

    An immunochemical approach was used to detect the expression of putative guanine nucleotide-binding proteins (G-proteins), arrestin, and nucleoside diphosphate kinases during wheat (Triticum aestivum) tissue culture initiated from immature embryos. Both the soluble and membrane extracts from the immature embryos revealed bands of 58, 40, and 16 kD with antibodies to G-protein (alpha subunit), arrestin, and nucleoside diphosphate kinase, respectively. These proteins were overexpressed in vitro in both nonembryogenic callus and embryogenic cultures. An additional soluble protein (32 kD) was detected by anti-G alpha antibodies in cultured tissues but not in immature embryos, suggesting a possible function in cell multiplication. Moreover, somatic embryogenesis was associated with the appearance of a 29-kD protein reactive with anti-arrstin antibodies, both in soluble and membrane fractions. Tissue-cultured genetic stocks of Chinese Spring wheat, including the disomic, 36 ditelosomic, and 6 nullisomic-tetrasomic wheat lines, were used to ascertain the chromosomal location of the genes encoding the 29-kD arrestin-like protein. The lack of a signal with the nonembryogenic ditelosomic 3 D short chromosome arm line suggests that the 3 D long chromosome arm possesses at least one gene involved in the expression of the 29-kD protein. The putative role of the 29-kD protein in signal-transduction regulating embryogenesis is discussed. PMID:9085574

  8. Microfluidic transwell inserts for generation of tissue culture-friendly gradients in well plates

    PubMed Central

    Sip, Christopher G.; Bhattacharjee, Nirveek; Folch, Albert

    2015-01-01

    Gradients of biochemical molecules play a key role in many physiological processes such as axon growth, tissue morphogenesis, and trans-epithelium nutrient transport, as well as in pathophysiological phenomena such as wound healing, immune response, bacterial invasion, and cancer metastasis. In this paper, we report a microfluidic transwell insert for generating quantifiable concentration gradients in a user-friendly and modular format that is compatible with conventional cell cultures and with tissue explant cultures. The device is simply inserted into a standard 6-well plate, where it hangs self-supported at a distance of ~250 μm above the cell culture surface. The gradient is created by small microflows from the device, through an integrated track-etched porous membrane, into the cell culture well. The microfluidic transwell can deliver stable, quantifiable gradients over a large area with extremely low fluid shear stress to dissociated cells or tissue explants cultured independently on the surface of a 6-well plate. We used finite-element modeling to describe the porous membrane flow and molecular transport and to predict gradients generated by the device. Using the device, we applied a gradient of the chemotactic peptide N-Formyl-Met-Leu-Phe (fMLP) to a large population of HL-60 cells (a neutrophil cell line) and directly observed the migration with time-lapse microscopy. On quantification of the chemotactic response with an automated tracking algorithm, we found 74% of the cells moving towards the gradient. Additionally, the modular design and low fluid shear stress made it possible to apply gradients of growth factors and second messengers to mouse retinal explant cultures. With a simplified interface and well-defined gradients, the microfluidic transwell device has potential for broad applications to gradient-sensing biology. PMID:24225908

  9. Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

    PubMed Central

    Fayazi, Mehri; Salehnia, Mojdeh; Ziaei, Saeideh

    2016-01-01

    Background: The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage. Methods: Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105. Results: 11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. Conclusion: The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data. PMID:26568058

  10. Functional enhancement of chitosan and nanoparticles in cell culture, tissue engineering, and pharmaceutical applications

    PubMed Central

    Gao, Wenjuan; Lai, James C. K.; Leung, Solomon W.

    2012-01-01

    As a biomaterial, chitosan has been widely used in tissue engineering, wound healing, drug delivery, and other biomedical applications. It can be formulated in a variety of forms, such as powder, film, sphere, gel, and fiber. These features make chitosan an almost ideal biomaterial in cell culture applications, and cell cultures arguably constitute the most practical way to evaluate biocompatibility and biotoxicity. The advantages of cell cultures are that they can be performed under totally controlled environments, allow high throughput functional screening, and are less costly, as compared to other assessment methods. Chitosan can also be modified into multilayer composite by combining with other polymers and moieties to alter the properties of chitosan for particular biomedical applications. This review briefly depicts and discusses applications of chitosan and nanoparticles in cell culture, in particular, the effects of chitosan and nanoparticles on cell adhesion, cell survival, and the underlying molecular mechanisms: both stimulatory and inhibitory influences are discussed. Our aim is to update the current status of how nanoparticles can be utilized to modify the properties of chitosan to advance the art of tissue engineering by using cell cultures. PMID:22934070

  11. Three-dimensional hydrogel cell culture systems for modeling neural tissue

    NASA Astrophysics Data System (ADS)

    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

  12. Survival of isolated human islets of Langerhans maintained in tissue culture.

    PubMed Central

    Andersson, A; Borg, H; Groth, C G; Gunnarsson, R; Hellerström, C; Lundgren, G; Westman, J; Ostman, J

    1976-01-01

    Transplantation of human pancreatic islets to diabetic patients may require that donor islets be kept viable in vitro for extended time periods before transfer to the recipient. We have maintained isolated pancreatic islets obtained from the human cadaveric pancreas in tissue culture for 1-3 wk, after which we studied the structure and function of the islets. Electron micrographs of the cultured islets showed a satisfactory preservation of both beta-cells and alpha 2-cells. After culture for 1 wk, the islet oxygen uptake proceeded at a constant rate at a low glucose concentration (3.3 mM) and was significantly enhanced by raising the glucose concentration to 16.7 mM. Likewise, after culture for 1 wk, the islets responded with an increased insulin release when exposed to 16.7 mM glucose with or without added theophylline (10 mM). Islets cultured for 1-3 wk were able to incorporate [3H]leucine into proinsulin, as judged by gel filtration of acid-alcohol extracts. Glucagon release from the cultured islets was reduced significantly by 16.7 mM glucose alone, but stimulated by glucose (16.7 mM) plus theophylline (10 MM). It is concluded that viable pancreatic islets can be isolated from the pancreas of adult human donors and maintained in tissue culture for at least 1 wk without loss of the specific functions of the alpha 2- and beta-cells. It remains to be established whether such islets will survive and remain functionally competent after transplantation to human recipients. Images PMID:770504

  13. Enhanced anthocyanins and resveratrol production in Vitis vinifera cell suspension culture by indanoyl-isoleucine, N-linolenoyl-L-glutamine and insect saliva.

    PubMed

    Cai, Zhenzhen; Knorr, Dietrich; Smetanska, Iryna

    2012-01-01

    The effects of two synthetic elicitor indanoyl-isoleucine (In-Ile), N-linolenoyl-L-glutamine (Lin-Gln) and one biotic elicitor insect saliva (from Manduca sexta larvae) on plant cell cultures with respect to the induction of secondary metabolite production were investigated. Stimulated production of secondary metabolites, particularly anthocyanins in plant cells and phenolic acids in culture medium, was studied by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. In the treatments with In-Ile, the production of anthocyanins was enhanced 2.6-fold. In-Ile, Lin-Gln and saliva significantly elevated the accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol. The used elicitors did not suppress cell growth. Secondary metabolites were differently responsive to elicitation. 3-O-glucosyl-resveratrol was the predominant phenolic acid in V. vinifera cell culture, and its production was significantly stimulated by saliva, with 7.0-fold of the control level 24 h after treatment. The production of 4-(3,5-dihydroxy-phenyl)-phenol was significantly stimulated by In-Ile with 6.4-fold of the control level 24 h after treatment. PMID:22133437

  14. Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

    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.

  15. Effect of Selected Polysaccharide-Producing Soil Bacteria on Hyperhydricity Control in Oregano Tissue Cultures

    PubMed Central

    Ueno, K.; Shetty, K.

    1997-01-01

    Hyperhydricity, or vitrification, is a physiological malformation affecting tissue culture-generated plants. This malformation is associated with excessive hydration and poor lignification and results in poor regeneration of plants. We have tested hyperhydricity prevention in oregano by several nonspecific polysaccharide-producing rhizosphere bacteria. Among these bacteria, Pseudomonas mucidolens and another Pseudomonas sp. prevented hyperhydricity and improved acclimation of oregano clones. These two bacteria have more advantages for commercial applications than Pseudomonas strains isolated previously from oregano. PMID:16535525

  16. [Penetration of polyene antibiotics into human embryonic kidney tissue cell cultures].

    PubMed

    Kravchenko, L S; Sokolov, V N; Vaĭnshteĭn, V A; Diment, A V; Tereshin, I M

    1977-12-01

    Penetration of 14C-amphotericin AM-2 into the cells of the tissue culture of the human embryon kidneys was studied by means of light autoradiography after incubation with the antibiotic. Microscopic examination of the autographs of the cell slices revealed the presence of the radioactive label in the cytoplasm and nucleoplasm of the cells. The revealed intracellular localization of the label was evident of the antibiotic penetration into the cells. PMID:596858

  17. Morphological Variants of Sindbis Virus Obtained from Infected Mosquito Tissue Culture Cells

    PubMed Central

    Brown, Dennis T.; Gliedman, Jeffrey B.

    1973-01-01

    Tissue-cultured Aedes albopictus cells infected with morphologically homogeneous Sindbis virus were found to produce progeny virions which could be divided into three classes based on size. The thickness of the envelope was constant on all three sizes of progeny virions suggesting that the variability in size rested with the viral nucleocapsid. It is suggested that the three classes of virions have icosahedral nucleocapsids composed of common subunits organized in decreasing triangulation numbers. Images PMID:4128381

  18. Length of time in tissue culture can affect the selected glyphosate resistance mechanism.

    PubMed

    Papanikou, Efstratia; Brotherton, Jeffrey E; Widholm, Jack M

    2004-02-01

    Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity. We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism. Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill. cultures but did occur in all 10 lines selected after 52 months. Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes. Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence. None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years. However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture. These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance. PMID:14566562

  19. Air exposure induced characteristics of dry eye in conjunctival tissue culture.

    PubMed

    Lin, Hui; Qu, Yangluowa; Geng, Zhixin; Li, Cheng; Wu, Huping; Dong, Nuo; Liu, Zuguo; Li, Wei

    2014-01-01

    There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures. PMID:24498087

  20. The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture.

    PubMed

    Takahashi, Masakazu; Tsujimoto, Kazuhisa; Yamada, Hideyuki; Takagi, Hiroshi; Nakamori, Shigeru

    2003-11-01

    Sericin is the silk protein that covers fibroin fibers and functions as a 'glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 microg sericin hydrolysate and 350 microg SerD (the peptide containing the two repetitive units) ml(-1), respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation. PMID:14677702

  1. A Transcriptome-Based Characterization of Habituation in Plant Tissue Culture1[W

    PubMed Central

    Pischke, Melissa S.; Huttlin, Edward L.; Hegeman, Adrian D.; Sussman, Michael R.

    2006-01-01

    For the last 50 years, scientists have recognized that varying ratios of the plant hormones cytokinin and auxin induce plant cells to form particular tissues: undifferentiated calli, shoot structures, root structures, or a whole plant. Proliferation of undifferentiated callus tissue, greening, and the formation of shoot structures are all cytokinin-dependent processes. Habituation refers to a naturally occurring phenomenon whereby callus cultures, upon continued passage, lose their requirement for cytokinin. Earlier studies of calli with a higher-than-normal cytokinin content indicate that overproduction of cytokinin by the culture tissues is a possible explanation for this acquired cytokinin independence. A transcriptome-based analysis of a well established habituated Arabidopsis (Arabidopsis thaliana) cell culture line was undertaken, to explore genome-wide expression changes underlying the phenomenon of habituation. Increased levels of expression of the cytokinin receptor CRE1, as well as altered levels of expression of several other genes involved in cytokinin signaling, indicated that naturally acquired deregulation of cytokinin-signaling components could play a previously unrecognized role in habituation. Up-regulation of several cytokinin oxidases, down-regulation of several known cytokinin-inducible genes, and a lack of regulation of the cytokinin synthases indicated that increases in hormone concentration may not be required for habituation. In addition, up-regulation of the homeodomain transcription factor FWA, transposon-related elements, and several DNA- and chromatin-modifying enzymes indicated that epigenetic changes contribute to the acquisition of cytokinin habituation. PMID:16489130

  2. Identification of neurotoxic cytokines by profiling Alzheimer’s disease tissues and neuron culture viability screening

    PubMed Central

    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.

    2015-01-01

    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. PMID:26564777

  3. Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells.

    PubMed

    Allen, T D; Rutherford, S A; Murray, S; Gardiner, F; Kiseleva, E; Goldberg, M W; Drummond, S P

    2007-01-01

    Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3-5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging. PMID:17546013

  4. Insect Allergy.

    PubMed

    Lee, Hobart; Halverson, Sara; Mackey, Regina

    2016-09-01

    Insect bites and stings are common. Risk factors are mostly associated with environmental exposure. Most insect bites and stings result in mild, local, allergic reactions. Large local reactions and systemic reactions like anaphylaxis are possible. Common insects that bite or sting include mosquitoes, ticks, flies, fleas, biting midges, bees, and wasps. The diagnosis is made clinically. Identification of the insect should occur when possible. Management is usually supportive. For anaphylaxis, patients should be given epinephrine and transported to the emergency department for further evaluation. Venom immunotherapy (VIT) has several different protocols. VIT is highly effective in reducing systemic reactions and anaphylaxis. PMID:27545732

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

    NASA Astrophysics Data System (ADS)

    Nolte, David D.

    2016-03-01

    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.

  6. Cryopreservation, Culture, and Transplantation of Human Fetal Mesencephalic Tissue into Monkeys

    NASA Astrophysics Data System (ADS)

    Redmond, D. E.; Naftolin, F.; Collier, T. J.; Leranth, C.; Robbins, R. J.; Sladek, C. D.; Roth, R. H.; Sladek, J. R.

    1988-11-01

    Studies in animals suggest that fetal neural grafts might restore lost neurological function in Parkinson's disease. In monkeys, such grafts survive for many months and reverse signs of parkinsonism, without attendant graft rejection. The successful and reliable application of a similar transplantation procedure to human patients, however, will require neural tissue obtained from human fetal cadavers, with demonstrated cellular identity, viability, and biological safety. In this report, human fetal neural tissue was successfully grafted into the brains of monkeys. Neural tissue was collected from human fetal cadavers after 9 to 12 weeks of gestation and cryopreserved in liquid nitrogen. Viability after up to 2 months of storage was demonstrated by cell culture and by transplantation into monkeys. Cryopreservation and storage of human fetal neural tissue would allow formation of a tissue bank. The stored cells could then be specifically tested to assure their cellular identity, viability, and bacteriological and virological safety before clinical use. The capacity to collect and maintain viable human fetal neural tissue would also facilitate research efforts to understand the development and function of the human brain and provide opportunities to study neurological diseases.

  7. Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.

    PubMed

    Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C

    2015-01-01

    Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers. PMID:25741927

  8. Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

    PubMed Central

    Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V.; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C.

    2015-01-01

    Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers. PMID:25741927

  9. Culture environment-induced pluripotency of SACK-expanded tissue stem cells.

    PubMed

    Paré, Jean-François; Sherley, James L

    2011-01-01

    Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs) have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK) acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing. PMID:22523467

  10. Free Amino Acid Contents of Stem and Phylloxera Gall Tissue Cultures of Grape 1

    PubMed Central

    Warick, R. P.; Hildebrandt, A. C.

    1966-01-01

    Free amino acid constituents were determined of grape stem and Phylloxera leaf gall callus in tissue culture. Fast, medium and slow growing single cell clones of, respectively, stem and gall origins were grown on a mineral salt-sucrose medium supplemented with coconut milk and α-naphthaleneacetic acid. Stem and gall clones showed qualitative similarities and quantitative variations in the amino acids and nitrogenous constituents. Nineteen amino acids, glucosamine, ethanolamine, sarcosine, methionine sulfoxides and ammonia were identified. Two free polypeptides accounted for over 30% of the amino compounds in the stem and gall callus tissues which were not found in the intact plant parts. Stem clones of different growth rates grown on agar showed generally an excess of amino acid constituents over gall tissues of similar growth rates, except for the free polypeptides. Fast growing stem clones grown on agar medium contained lower amounts of certain amino acids than the fast growing gall clones, but when grown in liquid medium they contained higher amounts of these acids than the gall clones. The total and nonsoluble nitrogen of stem clones were higher than in the gall clones. Tissue cultures differed from the original plant parts with respect to their free polypeptides and high amino acid contents. Images PMID:16656290

  11. Free amino Acid contents of stem and phylloxera gall tissue cultures of grape.

    PubMed

    Warick, R P; Hildebrandt, A C

    1966-04-01

    Free amino acid constituents were determined of grape stem and Phylloxera leaf gall callus in tissue culture. Fast, medium and slow growing single cell clones of, respectively, stem and gall origins were grown on a mineral salt-sucrose medium supplemented with coconut milk and alpha-naphthaleneacetic acid. Stem and gall clones showed qualitative similarities and quantitative variations in the amino acids and nitrogenous constituents. Nineteen amino acids, glucosamine, ethanolamine, sarcosine, methionine sulfoxides and ammonia were identified. Two free polypeptides accounted for over 30% of the amino compounds in the stem and gall callus tissues which were not found in the intact plant parts. Stem clones of different growth rates grown on agar showed generally an excess of amino acid constituents over gall tissues of similar growth rates, except for the free polypeptides. Fast growing stem clones grown on agar medium contained lower amounts of certain amino acids than the fast growing gall clones, but when grown in liquid medium they contained higher amounts of these acids than the gall clones. The total and nonsoluble nitrogen of stem clones were higher than in the gall clones. Tissue cultures differed from the original plant parts with respect to their free polypeptides and high amino acid contents. PMID:16656290

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

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. )

    1990-08-01

    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.

  13. In Vitro Culture of Subanguina picridis in Acroptilon repens Callus, Excised Roots, and Shoot Tissues

    PubMed Central

    Ou, X.; Watson, A. K.

    1992-01-01

    The knapweed nematode Subanguina picridis is a foliar parasite that is of interest as a biological weed control agent of Russian knapweed. Attempts were made to culture the nematode in callus, excised roots and in shoots derived from roots of Russian knapweed. In callus tissues, the nematode developed from second-stage juvenile to adult but failed to reproduce; it developed only to the fourth stage in excised roots. However, S. picridis was successfully cultured in vitro in shoots derived from roots. The nematode induced galls on the leaves, petioles, and shoot apices and developed and reproduced inside the galls. Gibberellic acid increased the development rate of the nematode and promoted the formation of males. This is the first gnotobiotic culture of a nematode used for biological weed control. PMID:19283224

  14. Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents

    PubMed Central

    Kureshi, Alvena K; Dziasko, Marc; Funderburgh, James L; Daniels, Julie T

    2015-01-01

    Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease. PMID:26531048

  15. The structure of tissue on cell culture-extracted thyroglobulin is independent of its iodine content.

    PubMed

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

    1987-02-01

    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. PMID:3556752

  16. Culture and recovery of macrophages and cell lines from tissue culture-treated and -untreated plastic dishes.

    PubMed

    Fleit, S A; Fleit, H B; Zolla-Pazner, S

    1984-03-30

    Macrophages can be separated from other cell types by their ability to readily attach and spread on glass or on plastic surfaces which are treated for optimal growth of cultured cells (tissue culture-treated plastic). To detach macrophages from these surfaces, techniques must be used which require prior preparation of special flasks or vessels, utilize expensive equipment, are time-consuming and almost uniformly require that the macrophages be exposed to various chemicals. We now report that macrophages can be enriched and recovered efficiently after attachment to disposable polystyrene bacteriologic petri dishes simply by gentle scraping with a rubber policeman. In this paper we compare this method to others currently in use in which resident peritoneal cells, peritoneal exudate cells or cells from bone marrow-derived cultures are detached from treated dishes using cold shock, chelating agents and lidocaine. In all studies, advantages were noted when cells were incubated in untreated dishes and detached by gentle scraping. In addition, untreated dishes supported the growth of adherent cell lines IC-21 and L929B and yielded large numbers of cells, with high viability, which were easily harvested. PMID:6423730

  17. Colorectal cancer derived organotypic spheroids maintain essential tissue characteristics but adapt their metabolism in culture

    PubMed Central

    2014-01-01

    Background Organotypic tumor spheroids, a 3D in vitro model derived from patient tumor material, preserve tissue heterogeneity and retain structural tissue elements, thus replicating the in vivo tumor more closely than commonly used 2D and 3D cell line models. Such structures harbour tumorigenic cells, as revealed by xenograft implantation studies in animal models and maintain the genetic makeup of the original tumor material. The aim of our work was a morphological and proteomic characterization of organotypic spheroids derived from colorectal cancer tissue in order to get insight into their composition and associated biology. Results Morphological analysis showed that spheroids were of about 250 μm in size and varied in structure, while the spheroid cells differed in shape and size and were tightly packed together by desmosomes and tight junctions. Our proteomic data revealed significant alterations in protein expression in organotypic tumor spheroids cultured as primary explants compared to primary colorectal cancer tissue. Components underlying cellular and tissue architecture were changed; nuclear DNA/ chromatin maintenance systems were up-regulated, whereas various mitochondrial components were down-regulated in spheroids. Most interestingly, the mesenchymal cells appear to be substantial component in such cellular assemblies. Thus the observed changes may partly occur in this cellular compartment. Finally, in the proteomics analysis stem cell-like characteristics were observed within the spheroid cellular assembly, reflected by accumulation of Alcam, Ctnnb1, Aldh1, Gpx2, and CD166. These findings were underlined by IHC analysis of Ctnnb1, CD24 and CD44, therefore warranting closer investigation of the tumorigenic compartment in this 3D culture model for tumor tissue. Conclusions Our analysis of organotypic CRC tumor spheroids has identified biological processes associated with a mixture of cell types and states, including protein markers for mesenchymal

  18. Insect immunology and hematopoiesis.

    PubMed

    Hillyer, Julián F

    2016-05-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and activate effector pathways. Among the immune signaling pathways are the Toll, Imd, Jak/Stat, JNK, and insulin pathways. Activation of these and other pathways leads to pathogen killing via phagocytosis, melanization, cellular encapsulation, nodulation, lysis, RNAi-mediated virus destruction, autophagy and apoptosis. This review details these and other aspects of immunity in insects, and discusses how the immune and circulatory systems have co-adapted to combat infection, how hemocyte replication and differentiation takes place (hematopoiesis), how an infection prepares an insect for a subsequent infection (immune priming), how environmental factors such as temperature and the age of the insect impact the immune response, and how social immunity protects entire groups. Finally, this review highlights some underexplored areas in the field of insect immunobiology. PMID:26695127

  19. Insect Keepers

    ERIC Educational Resources Information Center

    Moore, Virginia J.; Chessin, Debby A.; Theobald, Becky

    2010-01-01

    Insects are fascinating creatures--especially when you and your students get up close and personal with them! To that end, the authors facilitated an inquiry-based investigation with an emphasis on identification of the different types of insects found in the school yard, their characteristics, their habitat, and what they eat, while engaging the…

  20. Incredible Insects.

    ERIC Educational Resources Information Center

    Braus, Judy, Ed.

    1989-01-01

    Ranger Rick's NatureScope is a creative education series dedicated to inspiring in children an understanding and appreciation of the natural world while developing the skills they will need to make responsible decisions about the environment. Contents are organized into the following sections: (1) "What Makes an Insect an Insect?," including…

  1. Tissue Inhibitor of Metalloproteinase-2 Suppresses Collagen Synthesis in Cultured Keloid Fibroblasts

    PubMed Central

    Dohi, Teruyuki; Aoki, Masayo; Ogawa, Rei; Akaishi, Satoshi; Shimada, Takashi; Okada, Takashi; Hyakusoku, Hiko

    2015-01-01

    Background: Keloids are defined as a kind of dermal fibroproliferative disorder resulting from the accumulation of collagen. In the remodeling of extracellular matrix, the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is as critical as the proper production of extracellular matrix. We investigate the role of TIMPs and MMPs in the pathogenesis of keloids and examine the therapeutic potential of TIMP-2. Methods: The expression of TIMPs and MMPs in most inflamed parts of cultured keloid fibroblasts (KFs) and peripheral normal skin fibroblasts (PNFs) in the same individuals and the reactivity of KFs to cyclic mechanical stretch were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (n = 7). To evaluate the effect of treating KFs with TIMP-2, collagen synthesis was investigated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and microscopic analysis was used to examine the treatment effects of TIMP-2 on ex vivo cultures of keloid tissue (n = 6). Results: TIMP-2 was downregulated in cultured KFs compared with PNFs in the same individuals, and the reduction in TIMP-2 was exacerbated by cyclic mechanical stretch. Administration of TIMP-2 (200 or 300 ng/mL) significantly suppressed expression of Col1A2 and Col3A1 mRNA and collagen type I protein in KFs. TIMP-2 also significantly reduced the skin dermal and collagen bundle thickness in ex vivo cultures of keloid tissue. Conclusion: These results indicated that downregulation of TIMP-2 in KFs is a crucial event in the pathogenesis of keloids, and the TIMP-2 would be a promising candidate for the treatment of keloids. PMID:26495233

  2. Well plate-based perfusion culture device for tissue and tumor microenvironment replication.

    PubMed

    Zhang, W; Gu, Y; Hao, Y; Sun, Q; Konior, K; Wang, H; Zilberberg, J; Lee, W Y

    2015-07-01

    There are significant challenges in developing in vitro human tissue and tumor models that can be used to support new drug development and evaluate personalized therapeutics. The challenges include: (1) working with primary cells which are often difficult to maintain ex vivo, (2) mimicking native microenvironments from which primary cells are harvested, and (3) the lack of culture devices that can support these microenvironments to evaluate drug responses in a high-throughput manner. Here we report a versatile well plate-based perfusion culture device that was designed, fabricated and used to: (1) ascertain the role of perfusion in facilitating the expansion of human multiple myeloma cells and evaluate drug response of the cells, (2) preserve the physiological phenotype of primary murine osteocytes by reconstructing the 3D cellular network of osteocytes, and (3) circulate primary murine T cells through a layer of primary murine intestine epithelial cells to recapitulate the interaction of the immune cells with the epithelial cells. Through these diverse case studies, we demonstrate the device's design features to support: (1) the convenient and spatiotemporal placement of cells and biomaterials into the culture wells of the device; (2) the replication of tissues and tumor microenvironments using perfusion, stromal cells, and/or biomaterials; (3) the circulation of non-adherent cells through the culture chambers; and (4) conventional tissue and cell characterization by plate reading, histology, and flow cytometry. Future challenges are identified and discussed from the perspective of manufacturing the device and making its operation for routine and wide use. PMID:26021852

  3. Assessment of Tissue Viability Following Electroosmotic Push–Pull Perfusion from Organotypic Hippocampal Slice Cultures

    PubMed Central

    2013-01-01

    We have developed a novel sampling technique that allows both introduction and removal of fluid from the extracellular space of living tissue. This method is based on the fluidics of push–pull perfusion but flow is driven by electroosmosis. We have applied this method to organotypic hippocampal cultures. A source capillary is inserted into the tissue and a collection capillary is in contact with the tissue surface through a thin layer of fluid. A voltage is applied across the proximal ends of source and collection capillary. In the applied field, fluid will move from source, into the tissue, and then be collected. In this process, damage to cells may occur. To understand better what sampling conditions influence damage most, we tested various sampling geometries and applied voltages, quantifying damage 16–24 h later using propidium iodide as a cell death marker. We found that damage correlates with both voltage drop and power dissipated in the tissue, but that voltage drop is a better indicator of damage when comparing models in which capillary arrangement and length are different. PMID:23639590

  4. Concise Review: Comparison of Culture Membranes Used for Tissue Engineered Conjunctival Epithelial Equivalents

    PubMed Central

    Eidet, Jon Roger; Dartt, Darlene A.; Utheim, Tor Paaske

    2015-01-01

    The conjunctival epithelium plays an important role in ensuring the optical clarity of the cornea by providing lubrication to maintain a smooth, refractive surface, by producing mucins critical for tear film stability and by protecting against mechanical stress and infectious agents. A large number of disorders can lead to scarring of the conjunctiva through chronic conjunctival inflammation. For controlling complications of conjunctival scarring, surgery can be considered. Surgical treatment of symblepharon includes removal of the scar tissue to reestablish the deep fornix. The surgical defect is then covered by the application of a tissue substitute. One obvious limiting factor when using autografts is the size of the defect to be covered, as the amount of healthy conjunctiva is scarce. These limitations have led scientists to develop tissue engineered conjunctival equivalents. A tissue engineered conjunctival epithelial equivalent needs to be easily manipulated surgically, not cause an inflammatory reaction and be biocompatible. This review summarizes the various substrates and membranes that have been used to culture conjunctival epithelial cells during the last three decades. Future avenues for developing tissue engineered conjunctiva are discussed. PMID:26690486

  5. Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine.

    PubMed

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

    2016-05-01

    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. PMID:27006260

  6. Prolonged survival of virulent Treponema pallidum (Nichols strain) in cell-free and tissue culture systems.

    PubMed

    Fieldsteel, A H; Becker, F A; Stout, J G

    1977-10-01

    Survival of Treponema pallidum was found to be prolonged in the presence of tissue culture. Of the 12 cultures studied, cottontail rabbit epithelium (Sf1Ep) supported T. pallidum for the longest time. In horizontal Leighton tubes with reduced medium and an atmosphere of 5% CO2 in N2, the 50% survival time (ST50) was 5 to 6 days for treponemes associated with monolayers of Sf1Ep cells. Comparable cell-free tubes had ST50 values of less than 4 days. In vertical Leighton tubes containing 6 ml of prereduced medium incubated aerobically, gradients of O2 tension and redox potential were established. Attachment and survival of T. pallidum were greatest at a depth of about 10 to 20 mm. Motility was between 70 and 95% in this area throughout the first 14 days of incubation. Occasionally, greater than 50% motility was observed for as long as 21 days. The redox potential and O2 tension in the optimal area of gradient cultures were reproduced by adjusting the medium depth in a shell vial culture system containing cells on a horizontal cover slip. Treponemes associated with the cell monolayer in both gradient and shell vial cultures were still virulent after 21 days in vitro. The dilution of testis extract and the concentration of T. pallidum were found to be important factors in survival of T. pallidum. PMID:332639

  7. Multilineage co-culture of adipose-derived stem cells for tissue engineering.

    PubMed

    Zhao, Yimu; Waldman, Stephen D; Flynn, Lauren E

    2015-07-01

    Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue-engineering strategies with adipose-derived stem cells (ASCs), the focus of this study was on developing indirect co-culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co-culture with each of the mature cell populations was shown to successfully induce or enhance lineage-specific differentiation of the ASCs. In general, a more homogeneous but lower-level differentiation response was observed in co-culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non-canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF-1 and DKK-1 on multilineage differentiation in co-culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co-culture with the mature cell populations. PMID:23135884

  8. Three-dimensional culture model for analyzing crosstalk between adipose tissue and hepatocytes.

    PubMed

    Nishijima-Matsunobu, Aki; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Fujimoto, Kazuma; Toda, Shuji

    2013-06-01

    Systemic adipose tissue is involved in the pathophysiology of obesity-associated liver diseases. However, a method has not been established for analyzing the direct interaction between adipose tissue and hepatocytes. We describe a useful three-dimensional model comprising a collagen gel coculture system in which HepG2 hepatocytes are cultured on a gel layer with visceral adipose tissue fragments (VAT) or subcutaneous tissue samples (SAT). Male adipose tissues were obtained from 5-week-old Wistar rats and human autopsy cases. Cellular behavior was analyzed by electron microscopy, immunohistochemistry, Western blot, real-time reverse transcription plus the polymerase chain reaction and enzyme-linked immunosorbent assay. VAT significantly promoted lipid accumulation and apoptosis in HepG2 cells and suppressed their growth and differentiation compared with SAT. VAT produced higher concentrations of fatty acids (palmitate, oleate, linoleate) than SAT. HepG2 cells significantly decreased the production of these fatty acids in VAT. Only HepG2 cells treated with 250 μM palmitate replicated VAT-induced apoptosis. Neither VAT nor SAT affected lipotoxicity-associated signals of nuclear factor kappa B, c-Jun N-terminal kinase and inositol requiring enzyme-1α in HepG2 cells. HepG2 cells never affected adiponectin, leptin, or resistin production in VAT and SAT. The data indicate that our model actively creates adipose tissue and HepG2 hepatocyte interactions, suggesting that (1) VAT plays more critical roles in hepatocyte lipotoxicity than SAT; (2) palmitate but not adipokines, is partly involved in the mechanisms of VAT-induced lipotoxicity; (3) HepG2 cells might inhibit fatty acid production in VAT to protect themselves against lipotoxicity. Our model should serve in studies of interactions between adipose tissue and hepatocytes and of the mechanisms in obesity-related lipotoxicity and liver diseases. PMID:23512139

  9. The Curious Connection Between Insects and Dreams

    PubMed Central

    Klein, Barrett A.

    2011-01-01

    A majority of humans spend their waking hours surrounded by insects, so it should be no surprise that insects also appear in humans’ dreams as we sleep. Dreaming about insects has a peculiar history, marked by our desire to explain a dream’s significance and by the tactic of evoking emotions by injecting insects in dream-related works of art, film, music, and literature. I surveyed a scattered literature for examples of insects in dreams, first from the practices of dream interpretation, psychiatry, and scientific study, then from fictional writings and popular culture, and finally in the etymology of entomology by highlighting insects with dream-inspired Latinate names. A wealth of insects in dreams, as documented clinically and culturally, attests to the perceived relevance of dreams and to the ubiquity of insects in our lives. PMID:26467945

  10. The Curious Connection Between Insects and Dreams.

    PubMed

    Klein, Barrett A

    2011-01-01

    A majority of humans spend their waking hours surrounded by insects, so it should be no surprise that insects also appear in humans' dreams as we sleep. Dreaming about insects has a peculiar history, marked by our desire to explain a dream's significance and by the tactic of evoking emotions by injecting insects in dream-related works of art, film, music, and literature. I surveyed a scattered literature for examples of insects in dreams, first from the practices of dream interpretation, psychiatry, and scientific study, then from fictional writings and popular culture, and finally in the etymology of entomology by highlighting insects with dream-inspired Latinate names. A wealth of insects in dreams, as documented clinically and culturally, attests to the perceived relevance of dreams and to the ubiquity of insects in our lives. PMID:26467945

  11. Morphological study of dynamic culture of thermosensitive collagen hydrogel in constructing tissue engineering complex.

    PubMed

    Huang, Lanfeng; Xu, Feixiang; Guo, Bin; Ma, Jianchao; Zhao, Jinsong

    2016-07-01

    ABSTACT The purpose of this study is to research the morphologies and functional characteristics of the cell-scaffold complex in vitro constructed under dynamic culture conditions. BMSCs were isolated from the long bones of Fischer344 rats, and performed in vitro amplification to the third generation as seed cells, together with thermosensitive collagen hydrogel (TCH) as cell adhesion matrix, and poly-L-lactic acid (PLLA) as scaffold, to construct cell-scaffold complex. The cell-scaffold complexes in the experiment group and the control group were then performed dynamic culture and static culture. After 7 d of in vitro culture, the complexes in the 2 groups were performed gross observation and SEM; meanwhile, the total DNA content in the complex was detected on D0,1,3, and 7 of culture. After cultured using these 2 ways, collagen could both wrap the PLLA scaffold, forming dense film-like structures on the PLLA surface. The total DNA contents in the cell-scaffold complex of the experiment group on D1,3, and 7 were significantly higher than the control group (P < 0.05). Compared with D0, the total DNA contents on D1,3, and 7 in both groups were gradually increased, but only the total DNA contents on D7 showed statistically significant difference than D0 (P < 0.05). TCH -PLLA fiber joint-constructed complex extracellular matrix had good biocompatibility, and dynamic culture could promote the distribution of BMSCs on the surface and inside the structure, thus promoting cell proliferation, so it could be used for the in vitro construction of tissue engineering complex. PMID:27459597

  12. Isolation of Naturally Occurring Viruses of the Murine Leukemia Virus Group in Tissue Culture

    PubMed Central

    Hartley, Janet W.; Rowe, Wallace P.; Capps, Worth I.; Huebner, Robert J.

    1969-01-01

    A tissue culture cell system for isolation and identification of members of the murine leukemia virus group (the complement fixation for murine leukemia test) was modified to permit the isolation of naturally occurring virus from leukemic and normal mice. The important factors for increasing the sensitivity of the test were the use of National Institutes of Health (NIH) strain Webster Swiss embryo cell cultures and the selection of rat-immune sera having complement-fixing antibodies to tissue culture antigens of both the Gross and FMR subgroups. In all, 163 strains of mouse leukemia virus, from 11 inbred mouse strains, have been isolated. Representative virus isolates were shown to possess the properties of the murine leukemia virus group; i.e., they were chloroform-sensitive, noncytopathic agents which replicated in mouse embryo tissue culture and produced group-reactive, complement-fixing antigen and budding C-type particles visible by electron microscopy. These viruses could serve as helpers in the rescue of Moloney sarcoma virus genome from non-producer hamster sarcoma cells, yielding pseudotypes. All of the 19 field isolates tested were neutralized by Gross passage A antiserum but not by potent antisera to the Moloney, Rauscher, and Friend strains. Virus was recovered regularly from embryos and from the plasma and spleen of adult mice of high leukemic strains. In low leukemic mouse strains, different patterns of virus detection were observed. In C3H/He mice, virus was occasionally present in embryos and was found in 40% of adult spleens. BALB/c mice were virus-negative as fetuses or weanlings, but spleens of more than half of the mice over 6 months of age yielded virus. NIH mice have never yielded virus. In reciprocal matings between AKR and BALB/c mice, virus recovery from embryos was maternally determined. The development of tissue culture isolation procedures made possible for the first time the application of classical infectious disease methods to the

  13. Studies on the Nature of Receptors Involved in Attachment of Tissue Culture Cells to Mycoplasmas

    PubMed Central

    Manchee, R. J.; Taylor-Robinson, D.

    1969-01-01

    Several mycoplasmas, from avian and mammalian sources, growing in the form of colonies on agar and sheets attached to plastic dishes, were tested for their ability to adsorb tissue culture cells in suspension. HeLa cells adsorbed to the majority of mycoplasmas tested; adsorption occurred to the sheets and not to the colonies of some mycoplasmas. Other tissue cells, in primary culture and of diploid origin, adsorbed also. The mechanism of adsorption of HeLa cells to 4 mycoplasmas was examined by treating the cells and mycoplasmas in various ways and then testing for adsorption. N-acetyl neuraminic acid residues on the tissue cells were responsible for adsorption to M. gallisepticum and M. pneumoniae. The receptors for M. hominis and M. salivarium were probably not of this kind since treatment of the cells with purified neuraminidase did not influence adsorption. However, the cell receptors for these mycoplasmas were associated with protein because they were inactivated by proteolytic enzymes and by formalin. The cell receptors for M. hominis were more heat stable than those for the other mycoplasmas. From the aspect of the mycoplasma membrane, in no instance did neuraminidase treatment affect adsorption. On the other hand, various experiments suggested that protein components of the mycoplasma membrane were involved. The significance of these findings is discussed. PMID:5773147

  14. Dynamic Culture Conditions to Generate Silk-Based Tissue-Engineered Vascular Grafts

    PubMed Central

    Zhang, Xiaohui; Wang, Xiuli; Keshav, Vinny; Wang, Xiaoqin; Johanas, Jacqueline; Leisk, Gary; Kaplan, David L

    2009-01-01

    Tissue engineering is an alternative approach for the preparation of small-diameter (<6 mm) vascular grafts due to the potential to control thrombosis, anastomotic cellular hyperplasia and matrix production. This control also requires the maintenance of graft patency in vivo, appropriate mechanical properties and the formation of a functional endothelium. As a first step in generating such tissue-engineered vascular grafts (TEVG), our objective was to develop a tissue-engineered construct that mimicked the structure of blood vessels using tubular electrospun silk fibroin scaffolds (ESFS) with suitable mechanical properties. Human coronary artery smooth muscle cells (HCASMCs) and human aortic endothelial cells (HAECs) were sequentially seeded onto the luminal surface of the tubular scaffolds and cultivated under physiological pulsatile flow. The results demonstrated that TEVGs under dynamic flow conditions had better outcome than static culture controls in terms of cell proliferation and alignment, ECM production and cell phenotype based on transcript and protein level assessments. The metabolic activity of HCASMCs present in the TEGs indicated the advantage of dynamic flow over static culture in effective nutrient and oxygen distribution to the cells. A matrigel coating as a basement membrane mimic for ECM significantly improved endothelium coverage and retention under physiological shear forces. The results demonstrate the successful integration of vascular cells into silk electrospun tubular scaffolds as a step toward the development of a TEVG similar to native vessels in terms of vascular cell outcomes and mechanical properties. PMID:19232717

  15. Thick-tissue bioreactor as a platform for long-term organotypic culture and drug delivery

    PubMed Central

    Markov, Dmitry A.; Lu, Jenny Q.; Samson, Philip C.; Wikswo, John P.; McCawley, Lisa J.

    2013-01-01

    We have developed a novel, portable, gravity-fed, microfluidics-based platform suitable for optical interrogation of long-term organotypic cell culture. This system is designed to provide convenient control of cell maintenance, nutrients, and experimental reagent delivery to tissue-like cell densities housed in a transparent, low-volume microenvironment. To demonstrate the ability of our Thick-Tissue Bioreactor (TTB) to provide stable, long-term maintenance of high-density cellular arrays, we observed the morphogenic growth of human mammary epithelial cell lines, MCF-10A and their invasive variants, cultured under three-dimensional (3D) conditions inside our system. Over the course of 21 days, these cells typically develop into hollow “mammospheres” if cultured in standard 3D Matrigel. This complex morphogenic process requires alterations in a variety of cellular functions, including degradation of extracellular matrix that is regulated by cell-produced matrix proteinases. For our “drug” delivery testing and validation experiments we have introduced proteinase inhibitors into the fluid supply system, and we observed both reduced proteinase activity and inhibited cellular morphogenesis. The size inhibition results correlated well with the overall proteinase activities of the tested cells. PMID:22964798

  16. Cardiac Cell Culture Model (CCCM) as a Left Ventricle Mimic for Cardiac Tissue Generation

    PubMed Central

    Nguyen, Mai-Dung; Tinney, Joseph P.; Yuan, Fangping; Roussel, Thomas J.; El-Baz, Ayman; Giridharan, Guruprasad; Keller, Bradley B.; Sethu, Palaniappan

    2013-01-01

    A major challenge in cardiac tissue engineering is the delivery of hemodynamic mechanical cues that play a critical role in the early development and maturation of cardiomyocytes. Generation of functional cardiac tissue capable of replacing or augmenting cardiac function therefore requires physiologically relevant environments that can deliver complex mechanical cues for cardiomyocyte functional maturation. The goal of this work is the development and validation of a cardiac cell culture model (CCCM) microenvironment that accurately mimics pressure-volume changes seen in the left ventricle and to use this system to achieve cardiac cell maturation under conditions where mechanical loads such as pressure and stretch are gradually increased from the unloaded state to conditions seen in vivo. The CCCM platform, consisting of a cell culture chamber integrated within a flow loop was created to accomplish culture of 10 day chick embryonic ventricular cardiomyocytes subject to 4 days of stimulation (10 mm Hg, ~13% stretch at a frequency of 2 Hz). Results clearly show that CCCM conditioned cardiomyocytes accelerate cardiomyocyte structural and functional maturation in comparison to static unloaded controls as evidenced by increased proliferation, alignment of actin cytoskeleton, bundle-like sarcomeric α-actinin expression, higher pacing beat rate at lower threshold voltages and increased shortening. These results confirm the CCCM microenvironment can accelerate immature cardiac cell structural and functional maturation for potential cardiac regenerative applications. PMID:23952579

  17. Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture

    PubMed Central

    Kumar, Jyoti; Dixit, Shivendra Kumar; Kumar, Rajiv

    2015-01-01

    Aim: This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. Introduction: M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. Materials and Methods: A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. Results: All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence

  18. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture

    PubMed Central

    Reda, A.; Hou, M.; Winton, T.R.; Chapin, R.E.; Söder, O.; Stukenborg, J.-B.

    2016-01-01

    STUDY QUESTION Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no

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

    PubMed Central

    Ozbun, Michelle A.; Patterson, Nicole A.

    2014-01-01

    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 for growing differentiating epithelial tissues, whose structure and function mimics many important morphological and biochemical aspects of normal skin. The technique, pioneered by Asslineau and Pruniéras (Asselineau and Prunieras 1984) and modified by Kopan et al. (Kopan et al. 1987), 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, as well as 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

  20. Tissue Culture-Induced Heritable Genomic Variation in Rice, and Their Phenotypic Implications

    PubMed Central

    Gao, Yang; Liu, Ying; Wu, Ying; Bai, Yan; Zhang, Zhibin; Lin, Xiuyun; Dong, Yuzhu; Ou, Xiufang; Xu, Chunming; Liu, Bao

    2014-01-01

    Background Somaclonal variation generally occurs in plants regenerated from tissue culture. However, fundamental issues regarding molecular characteristics, mutation rates and mutation spectra of plant somatic variation as well as their phenotypic relevance have been addressed only recently. Moreover, these studies have reported highly discrepant results in different plant species and even in the same plant genotype. Methodology/principal findings We investigated heritable genomic variation induced by tissue culture in rice by whole genome re-sequencing of an extensively selfed somaclonal line (TC-reg-2008) and its wild type (WT) donor (cv. Hitomebore). We computed the overall mutation rate, single nucleotide polymorphisms (SNPs), small scale insertions/deletions (Indels) and mobilization of transposable elements (TEs). We assessed chromosomal distribution of the various types of genomic variations, tested correlations between SNPs and Indels, and examined concomitancy between TE activity and its cytosine methylation states. We also performed gene ontology (GO) analysis of genes containing nonsynonymous mutations and large-effect mutations, and assayed effects of the genomic variations on phenotypes under both normal growing condition and several abiotic stresses. We found that heritable somaclonal genomic variation occurred extensively in rice. The genomic variations distributed non-randomly across each of the 12 rice chromosomes, and affected a large number of functional genes. The phenotypic penetrance of the genomic variations was condition-dependent. Conclusions/significance Tissue culture is a potent means to generate heritable genetic variations in rice, which bear distinct difference at least in space (chromosomal distribution) from those occurred under natural settings. Our findings have provided new information regarding the mutation rate and spectrum as well as chromosomal distribution pattern of somaclonal variation in rice. Our data also suggest that

  1. Response of tobacco tissue cultures growing in contact with lunar fines.

    NASA Technical Reports Server (NTRS)

    Weete, J. D.; Walkinshaw, C. H.; Laseter, J. L.

    1973-01-01

    During the quarantine periods following each Apollo mission to the moon, various biological systems were placed in the presence of lunar material to determine if pathogenic agents were present. Although no detrimental effects resulted, various responses by the several plant systems tested were noted. One such response was the increased pigmentation observed in the callus tissue cultures of tobacco. Further investigations revealed that these tissues grown in the presence of lunar material resulted in as much as a 35% increase in total pigments while differences in fatty acid and sterol concentrations were also noted when compared to the controls. It is believed that these changes brought about by the lunar material can be attributed to a change in the nutritional environment caused by its dissolution.

  2. Direct Fluorescent Detection of a Polymethoxyflavone in Cell Culture and Mouse Tissue.

    PubMed

    Chen, Jingjing; Song, Mingyue; Wu, Xian; Zheng, Jinkai; He, Lili; McClements, David Julian; Decker, Eric; Xiao, Hang

    2015-12-16

    Convenient detection methods for bioactive food compounds and their metabolites in biological samples are needed to better understand their mechanism of actions. Herein, we developed a novel approach to directly monitor and visualize the distribution of 5,3',4'-tridemethylnobiletin (TDN), a unique polymethoxyflavone metabolite derived from citrus polymethoxyflavone, in biological samples such as cultured cells and mouse colonic tissues. Our results showed that a fluorescent conjugate could be formed between TDN and 2-aminoethyl diphenyl borate (DPBA) under simple reaction conditions, which was confirmed by both Raman spectroscopy and mass spectroscopy. We further demonstrated the application of DPBA-based conjugation reaction in the characterization of TDN in different biological samples including floating cells, adherent cells, and animal tissues. This is the first report demonstrating direct fluorescent detection of polymethoxyflavone in biological samples. PMID:26618604

  3. Genetic variability of somatic embryogenesis in tissue cultures of sugar beet breeding lines.

    PubMed

    Golovko, A

    2001-01-01

    Genetic variability of callus initiation and plant regeneration has been investigated among three sugar beet genotypes. It was found that TDZ has a genotype-independent effect on callus initiation and is responsible for more than a two-fold increase in the friable callus induction rate and more than a three-fold increase in the shoot regeneration rate from this callus. Along with the genotype-independent organogenesis, regeneration from callus occasionally went through the process of somatic embryogenesis in a highly genotype-specific manner. Despite fast and uncontrollable conversion of embryos to normal plants, it was possible to select and maintain repetitive embryogenic culture without loosing regeneration and root formation capabilities. Extensive experimenting with medium composition and culture conditions resulted in an optimal medium for maintenance of repetitive embryos. Comparing with BAP, low concentrations of TDZ provide higher level of adventitious shoot formation and do not induce vitrification of tissues. PMID:11944321

  4. The safety assessment of food ingredients derived from plant cell, tissue and organ cultures: a review.

    PubMed

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

    2015-06-01

    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. PMID:25624252

  5. Microfluidic Probe for Single-Cell Lysis and Analysis in Adherent Tissue Culture

    PubMed Central

    Lauffenburger, Douglas A.; Han, Jongyoon

    2014-01-01

    Single-cell analysis provides information critical to understanding key disease processes that are characterized by significant cellular heterogeneity. Few current methods allow single-cell measurement without removing cells from the context of interest, which not only destroys contextual information but also may perturb the process under study. Here we present a microfluidic probe that lyses single adherent cells from standard tissue culture and captures the contents to perform single-cell biochemical assays. We use this probe to measure kinase and housekeeping protein activities, separately or simultaneously, from single human hepatocellular carcinoma cells in adherent culture. This tool has the valuable ability to perform measurements that clarify connections between extracellular context, signals and responses, especially in cases where only a few cells exhibit a characteristic of interest. PMID:24594667

  6. Reconstruction of Auto-Tissue-Engineered Lamellar Cornea by Dynamic Culture for Transplantation: A Rabbit Model

    PubMed Central

    Duan, Haoyun; Wang, Xiaoran; Xiao, Jianhui; Duan, Hucheng; Li, Naiyang; Li, Chaoyang; Wan, Pengxia; Liu, Ying; Song, Yiyue; Zhou, Chenjing; Huang, Zheqian; Wang, Zhichong

    2014-01-01

    To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3−, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63−, ABCG2−). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function. PMID:24705327

  7. Reconstruction of auto-tissue-engineered lamellar cornea by dynamic culture for transplantation: a rabbit model.

    PubMed

    Wu, Zheng; Zhou, Qiang; Duan, Haoyun; Wang, Xiaoran; Xiao, Jianhui; Duan, Hucheng; Li, Naiyang; Li, Chaoyang; Wan, Pengxia; Liu, Ying; Song, Yiyue; Zhou, Chenjing; Huang, Zheqian; Wang, Zhichong

    2014-01-01

    To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3-, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63-, ABCG2-). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function. PMID:24705327

  8. Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

    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.

  9. Survival of cultured plant cells grafted into the subcutaneous tissue of rats (preliminary report).

    PubMed

    Lozoya, X; Madrazo, I; Guizar, G; Villarreal, M L; Grijalva, I; Salgado, H; Boijseauneau, E; Ibarra, A; Arias-Castro, C; Rodríguez-Mendiola, M A

    1995-01-01

    To evaluate the survival of plant tissue in an animal environment, cultured calli from a Mexican medicinal plant (Mimosa tenuiflora Poir.) were transplanted under sterile conditions into the subcutaneous tissue of rats. Microscopic studies of grafted areas were carried out at the 30th, 60th and 120th days after transplantation. Histological evidence of plant graft survival was found in specimens of all groups. during the first month of subcutaneous grafting a moderate inflammatory reaction around the callus was observed characterized by the presence of polymorphonuclear cells and some macrophages and the formation of a fibrous capsule. Nevertheless, the plant grafts remained viable and a decrease of the inflammatory reaction around the callus was observed in the specimens during the following months. In the fourth month specimens the formation of blood vessels inside the grafted plant tissue was observed. Once removed from rats, plant tissues showed high viability according to the fluorescein test. These calli were then transferred to the original in vitro medium showing growth capacity during the following weeks. These results demonstrate, for the first time, that cultivated cells of higher plants survive in an animal environment, suggesting the possibility to utilize pharmacologically active plant transplants in animals, a technique proposed here as inter-regni transplants. Further studies are required to explore this new field of research that opens numerous questions about plant-animal cellular interaction. PMID:7711454

  10. Biotransformation of tissue-specific hormone tibolone with fungal culture Trichothecium roseum

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    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.

  11. Analysis of laser-induced fluorescence spectra of in vitro plant tissue cultures.

    PubMed

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

    2007-04-10

    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. PMID:17384731

  12. Protection of cattle against rinderpest by intranasal immunisation with a dry powder tissue culture vaccine.

    PubMed

    Anderson, J; Fishbourne, E; Corteyn, A; Donaldson, A I

    2000-11-22

    Dry powder tissue culture rinderpest vaccine containing 10(2.5) TCID(50) of virus per dose administered intranasally to cattle induced high titre circulating antibody responses and protection against challenge with a virulent strain of rinderpest virus. A reduction in the dose of virus to 10(1.1) TCID(50) resulted in a failure to elicit detectable antibody responses and a lack of protection. Intranasal powder vaccine offers several advantages over conventional needle-administered aqueous rinderpest vaccine, including greater stability in the absence of a cold chain, reduced risk of 'needle transfer' of other microbial agents present in the vaccinated herd and lower cost. PMID:11115707

  13. Tissue Culture Study of The Medicinal Plant Leek (Allium Ampeloprasum L)

    PubMed Central

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

    2014-01-01

    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. PMID:25035862

  14. Tissue culture study of the medicinal plant leek (allium ampeloprasum L).

    PubMed

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

    2014-01-01

    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. PMID:25035862

  15. [THE REGULATING EFFECT OF DIPEPTIDES ON CELL PROLIFERATION IN NERVE TISSUE CULTURE IN MAMMALS AND ON ASSOCIATIVE LEARNING IN INSECTS].

    PubMed

    Chalisova, N I; Zachepilo, T G; Kamyshev, N G; Lopatina, N G

    2015-01-01

    The effect of dipeptides AspPro and AspSer and of their composing amino acids (asparagine acid--Asp, proline--Pro, serin--Ser) on the proliferative activity in the explants of cortex and subcortical structures of the rat brain and on the functional activity of CNS of the honeybee was studied. The square index defined as a proportion of the whole explant square to the square of its central zone was determined. The number of bees responded with the conditional reaction (proboscis extension in the direction to aromatized solution) after 1 min (short-term memory) and 180 min (long-term memory) was detected after single learning procedure. Both dipeptides, as well as the asparagine acid, stimulated an increase of the growth zone of the subcortical structure explants in rats and of the number of honeybees with retention of conditional reaction in the short-term/long-term memory independently of the effect of the second member of the dipeptide. The unidirectionality of the effect suggests the existence of common mechanisms of reception and signal transduction established during evolution that require the further study. PMID:26983279

  16. Tissue culture on a chip: Developmental biology applications of self-organized capillary networks in microfluidic devices.

    PubMed

    Miura, Takashi; Yokokawa, Ryuji

    2016-08-01

    Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long-term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self-organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology. PMID:27272910

  17. Plant Defense against Insect Herbivores

    PubMed Central

    Fürstenberg-Hägg, Joel; Zagrobelny, Mika; Bak, Søren

    2013-01-01

    Plants have been interacting with insects for several hundred million years, leading to complex defense approaches against various insect feeding strategies. Some defenses are constitutive while others are induced, although the insecticidal defense compound or protein classes are often similar. Insect herbivory induce several internal signals from the wounded tissues, including calcium ion fluxes, phosphorylation cascades and systemic- and jasmonate signaling. These are perceived in undamaged tissues, which thereafter reinforce their defense by producing different, mostly low molecular weight, defense compounds. These bioactive specialized plant defense compounds may repel or intoxicate insects, while defense proteins often interfere with their digestion. Volatiles are released upon herbivory to repel herbivores, attract predators or for communication between leaves or plants, and to induce defense responses. Plants also apply morphological features like waxes, trichomes and latices to make the feeding more difficult for the insects. Extrafloral nectar, food bodies and nesting or refuge sites are produced to accommodate and feed the predators of the herbivores. Meanwhile, herbivorous insects have adapted to resist plant defenses, and in some cases even sequester the compounds and reuse them in their own defense. Both plant defense and insect adaptation involve metabolic costs, so most plant-insect interactions reach a stand-off, where both host and herbivore survive although their development is suboptimal. PMID:23681010

  18. Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study

    PubMed Central

    Fox, Derek B.; Stoker, Aaron M.; Beatty, Mark; Cockrell, Mary; Janicek, John C.; Cook, James L.

    2014-01-01

    Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA

  19. New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles.

    PubMed

    Li, You; You, Li; Simmons, David Rabern; Bateman, Craig C; Short, Dylan P G; Kasson, Matthew T; Rabaglia, Robert J; Hulcr, Jiri

    2015-01-01

    Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and includes many notorious invasive pests. All ambrosia beetles studied to date cultivate ascomycotan fungi: early colonizers of recently killed trees with poor wood digestion. Beetles in the widespread genus Ambrosiodmus, however, colonize decayed wood. We characterized the mycosymbionts of three Ambrosiodmus species using quantitative culturing, high-throughput metabarcoding, and histology. We determined the fungi to be within the Polyporales, closely related to Flavodon flavus. Culture-independent sequencing of Ambrosiodmus minor mycangia revealed a single operational taxonomic unit identical to the sequences from the cultured Flavodon. Histological sectioning confirmed that Ambrosiodmus possessed preoral mycangia containing dimitic hyphae similar to cultured F. cf. flavus. The Ambrosiodmus-Flavodon symbiosis is unique in several aspects: it is the first reported association between an ambrosia beetle and a basidiomycotan fungus; the mycosymbiont grows as hyphae in the mycangia, not as budding pseudo-mycelium; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations. PMID:26367271

  20. New Fungus-Insect Symbiosis: Culturing, Molecular, and Histological Methods Determine Saprophytic Polyporales Mutualists of Ambrosiodmus Ambrosia Beetles

    PubMed Central

    Bateman, Craig C.; Short, Dylan P. G.; Kasson, Matthew T.; Rabaglia, Robert J.; Hulcr, Jiri

    2015-01-01

    Ambrosia symbiosis is an obligate, farming-like mutualism between wood-boring beetles and fungi. It evolved at least 11 times and includes many notorious invasive pests. All ambrosia beetles studied to date cultivate ascomycotan fungi: early colonizers of recently killed trees with poor wood digestion. Beetles in the widespread genus Ambrosiodmus, however, colonize decayed wood. We characterized the mycosymbionts of three Ambrosiodmus species using quantitative culturing, high-throughput metabarcoding, and histology. We determined the fungi to be within the Polyporales, closely related to Flavodon flavus. Culture-independent sequencing of Ambrosiodmus minor mycangia revealed a single operational taxonomic unit identical to the sequences from the cultured Flavodon. Histological sectioning confirmed that Ambrosiodmus possessed preoral mycangia containing dimitic hyphae similar to cultured F. cf. flavus. The Ambrosiodmus-Flavodon symbiosis is unique in several aspects: it is the first reported association between an ambrosia beetle and a basidiomycotan fungus; the mycosymbiont grows as hyphae in the mycangia, not as budding pseudo-mycelium; and the mycosymbiont is a white-rot saprophyte rather than an early colonizer: a previously undocumented wood borer niche. Few fungi are capable of turning rotten wood into complete animal nutrition. Several thousand beetle-fungus symbioses remain unstudied and promise unknown and unexpected mycological diversity and enzymatic innovations. PMID:26367271

  1. Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture.

    PubMed

    Krahn, Katy Nash; Bouten, Carlijn V C; van Tuijl, Sjoerd; van Zandvoort, Marc A M J; Merkx, Maarten

    2006-03-15

    Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-alpha1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-alpha1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-alpha1I probe (apparent K(d) values of 0.5 and 50 microM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-alpha1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods. PMID:16476406

  2. The Early History of Tissue Culture in Britain: The Interwar Years

    PubMed Central

    WILSON, DUNCAN

    2005-01-01

    SUMMARY The technique of tissue culture has, throughout the twentieth century, become a mainstay of biomedical research, and exists today as a celebrated scientific tool. However, an examination of its early history demonstrates that it was once contested, with professional opinion differing as to its value to science and medicine, and, crucially for the purposes of this article, considerable public awareness of its potential and perceived pitfalls. Here, the hitherto neglected situation in the early British history of tissue culture will be studied, with the focus being the work performed at the Strangeways Research Laboratory in Cambridge during the interwar years of the last century. Examination of the early life of this institution shows that scientists eager to stress the technique’s viability tapped into popular sentiment to overstress its potential, in a fashion reminiscent of earlier experimental biologists and their contemporary American counterparts. This ultimately backfired on British culturists as the press coverage of their work became incredibly sensationalist, and increasingly sinister in tone, and scientific fact and fantastical speculation became inseparable. PMID:16532064

  3. Effect of tissue culture storage on the in vivo survival of canine osteochondral allografts.

    PubMed

    Oates, K M; Chen, A C; Young, E P; Kwan, M K; Amiel, D; Convery, F R

    1995-07-01

    In vitro studies in our laboratory have shown that the biomechanical and biochemical characteristics of osteochondral grafts can be preserved for as long as 28 days under tissue culture conditions. This study represents an attempt to extend these results to an in vivo model. In adult mongrel dogs, either an autograft, a fresh allograft, or a stored allograft was placed in a standardized defect on the weight-bearing surface of the medial femoral condyle. The stored grafts were kept at 4 degrees C in tissue culture medium for 14 days prior to implantation. The animals were killed at 12 weeks. Cartilage from the contralateral knee served as a control. The modulus and permeability of the cartilage were assessed with confined compression creep tests. The collagen and glycosaminoglycan contents were measured, and the cartilage was analyzed histologically with hematoxylin and eosin and safranin O stains. Grossly, the cartilage appeared viable at harvest. The histologic results were similar in the treatment groups, with the same spectrum of mild degenerative changes being noted in each group. The glycosaminoglycan content was significantly less in the autograft group than in its control group and than in the fresh allograft group. The glycosaminoglycan content did not differ significantly between fresh and stored allografts. The collagen content, modulus, and permeability did not differ either between experimental and control groups or between graft types. Our results support the conclusion that osteochondral allografts can be stored for as many as 14 days without significantly affecting the results of the procedure. PMID:7674072

  4. Tissue culture correlational study of genetic cholangiopathy of autosomal recessive polycystic kidney disease.

    PubMed

    Nakanuma, Yasuni; Sato, Yasunori; Harada, Kenichi

    2013-01-01

    Cholangiocytes are epithelial cells that line the biliary tract and are also known as biliary epithelial cells (BECs). In vitro culture studies of BECs in correlation with tissue section examination may give us a comprehensive analysis of biliary tract diseases. Herein, we discuss genetic cholangiopathy of autosomal recessive polycystic kidney disease (ARPKD), mainly using a polycystic kidney (PCK) rat, an animal model of ARPKD. The hepatobiliary lesions in ARPKD patients (Caroli's disease and congenital hepatic fibrosis) and in PCK rats are speculated to be related to mutations to polycystic kidney and hepatic disease 1 (PKHD1) which have been recently demonstrated, though the exact causal relation between these mutations and hepatobiliary pathology remain to be clarified. Recently we clarified that BECs of PCK rat showed increased cell proliferation followed by irregular dilatation of intrahepatic bile ducts. We also identified the essential involvement of the MEK5-ERK5 pathway in the abnormal proliferation of BECs in the PCK rat. The degradation of laminin and type IV collagen (basal membrane components of bile ducts) was closely related to the biliary dysgenesis and cystogenesis in the PCK rats. BECs also showed mesenchymal phenotype followed by progressive portal tract fibrosis, indicating TGF-β1 may be involved in this acquisition of mesenchymal phenotype. Detailed tissue culture correlation studies of ARPKD and PCK rats are mandatory to evaluate the pathogenesis of this genetic cholangiopathy. PMID:23097114

  5. Additively Manufactured Device for Dynamic Culture of Large Arrays of 3D Tissue Engineered Constructs.

    PubMed

    Costa, Pedro F; Hutmacher, Dietmar W; Theodoropoulos, Christina; Gomes, Manuela E; Reis, Rui L; Vaquette, Cédryck

    2015-04-22

    The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine. PMID:25721231

  6. Suppression of Rous Sarcoma Virus Growth in Tissue Cultures by Mycoplasma orale

    PubMed Central

    Somerson, Norman L.; Cook, M. K.

    1965-01-01

    Somerson, Norman L. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), and M. K. Cook. Suppression of Rous sarcoma virus growth in tissue cultures by Mycoplasma orale. J. Bacteriol. 90:534–540. 1965.—An agent which produced cell destruction in human diploid and chick-embryo fibroblasts was isolated from WI-26 strain of human diploid fibroblasts and shown to be a mycoplasma. The multiplication of Rous sarcoma virus (RSV) and Rous associated virus (RAV) was inhibited in WI-26, WI-38, and chick-embryo fibroblasts infected with this mycoplasma. The mycoplasma isolate, designated strain 941, reacted strongly in the complement-fixation test with antiserum to Mycoplasma orale CH19299, an isolate obtained from the human oral cavity. The cytopathic effect of mycoplasma strain 941 could be eliminated by growing the mycoplasma on an artificial agar medium before inoculation into chick-embryo fibroblasts. Serial passage in chick-embryo fibroblasts restored the cytopathogenicity of the agar-grown mycoplasma. However, growth of RSV and RAV was inhibited by both the tissue culture-grown and the agar-grown 941 strain, and also by the CH19299 strain which did not produce any cytopathic effect. Images PMID:14329470

  7. Mapping QTLs for Tissue Culture Response in Soybean (Glycine max (L.) Merr.)

    PubMed Central

    Yang, Chao; Zhao, Tuanjie; Yu, Deyue; Gai, Junyi

    2011-01-01

    Quantitative trait loci (QTLs) that control the tissue culture response in soybean were detected by using 184 recombinant inbred lines (RILs) derived from two varieties: Kefeng No.1 and Nannong 1138-2. The molecular map consisting of 834 molecular markers using this population covered space 2307.83 cM of the genome throughout 24 linkage groups. The performance of tissue culture in soybean was evaluated by two indices: callus induction frequency (CIF) and somatic embryos initiation frequency (SEIF). They were expressed as the number of explants producing callus/ the number of total explants and the number of explants producing somatic embryos/ the number of total explants, respectively. The RIL lines showed continuous segregation for both indices. With the composite interval mapping (CIM) described in Windows QTL Cartographer Version 2.5, three quantitative trait loci (QTLs) were identified for the frequency of callus induction, on chromosomes B2 and D2, accounting for phenotypic variation from 5.84% to 16.60%; four QTLs on chromosome G were detected for the frequency of somatic embryos initiation and explained the phenotypic variation from 7.79% to 14.16%. The information of new QTLs identified in the present study will contribute to genetic improvement of regeneration traits with marker-assisted selection (MAS) in soybean. PMID:21952936

  8. A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.

    PubMed

    Macaya, Constanza C; Saavedra, Patricio E; Cepeda, Rodrigo E; Nuñez, Viviana A; Sarrazin, Andres F

    2016-01-01

    The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods. PMID:26739999

  9. Transferability of Trypanosoma cruzi from mixed human host infection to Triatoma infestans and from insects to axenic culture.

    PubMed

    Ortiz, Sylvia; Zulantay, Inés; Apt, Werner; Saavedra, Miguel; Solari, Aldo

    2015-02-01

    The etiologic agent of Chagas disease is Trypanosoma cruzi, a protozoan whose life cycle involves obligatory passage through vertebrate and invertebrate hosts in a series of stages. The aim of this study was to explore the transferability of mixed discrete typing units (DTUs) of T. cruzi present in chronic chagasic patients when passed through an invertebrate host during xenodiagnosis (XD) and then when transferred to axenic cultures to obtain T. cruzi isolates. DTUs of T. cruzi present in these two hosts and axenic cultures were identified by kDNA PCR amplification and subsequent hybridization with DTU-specific probes. Mixtures of Tc I, Tc II, Tc V and Tc VI DTUs were detected in blood samples. However as a result of XD and axenic cultures it was possible to identify mostly Tc V. We conclude that the transferability of an isolate of T.cruzi derived from mixed DTUs present in human blood depends upon the starved invertebrate host used for xenodiagnosis. PMID:25240699

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

    PubMed

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

    2016-09-21

    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. PMID:27492338

  11. In vitro bioartificial skin culture model of tissue rejection and inflammatory/immune mechanisms.

    PubMed

    Strande, L F; Foley, S T; Doolin, E J; Hewitt, C W

    1997-06-01

    We hypothesized that an in vitro bioartificial skin rejection model using living LSEs grown in tissue culture could be developed for the study of autologous, allogenic, and/or xenogeneic inflammatory/immune mechanisms and topical immunosuppressive drugs. Human fibroblasts were mixed with type 1 rat-tail collagen to form a matrix (4 to 5 days), on which human keratinocytes were seeded. After a keratinocyte monolayer formed, CT cultures were raised to the air-liquid interface for continued growth. In the REJ LSE model, immunocytes isolated from human blood were seeded on top of the NHEK monolayer at the time of air-lifting. Thickness measurements of the acellular keratin and keratinocyte layers, and nuclear/cytoplasmic ratios, in both CT and REJ were made using digital image analysis. Immunostaining with anticytokeratin demonstrated a viable, keratin-producing epidermal layer; staining with anti-TGF-beta suggested a role for this cytokine in the rejection or wound-healing process. The LSE appeared histologically similar to normal human epidermis. Immunocytes added to the REJ cultures caused an obvious rejection response and were clearly identifiable in the gels as CD45+ staining cells. The LSE model appears promising for the study of immune/inflammatory mechanisms, thermal injury, screening antirejection agents that might be applied topically and as an in vitro replacement for skin graft studies in animals. PMID:9193551

  12. N-acetyl muramyl dipeptide stimulation of bone resorption in tissue culture.

    PubMed Central

    Dewhirst, F E

    1982-01-01

    N-Acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), a structurally defined fragment of bacterial peptidoglycan, stimulated significant release of previously incorporated 45Ca from fetal rat bones in tissue culture over the concentration range of 0.1 to 10.0 micrograms/ml. MDP-Stimulated bone resorption was not inhibited by the addition of the prostaglandin synthetase inhibitor indomethacin to the culture medium. MDP was neither mitogenic for nor stimulated the release of osteoclast-activating factor from cultured human peripheral blood mononuclear cells. Thus, MDP-stimulated bone resorption in vitro is mediated by a mechanism which is not dependent upon prostaglandins or osteoclast-activating factor. 6-O-Stearoyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, a lipophilic analog of MDP, was slightly more potent than MDP. Two diastereomers of MDP, N-acetyl-muramyl-L-alanyl-L-isoglutamine and N-acetyl-muramyl-D-alanyl-D-isoglutamine, which are inactive as adjuvants, were at least 1,000 times less active than MDP in stimulating bone resorption. The stereochemical specificity for bone-resorptive activity paralleled that required for adjuvant activity, macrophage activation, and activation of the reticuloendothelial system. PMID:7054120

  13. In vitro culture of mantle tissue of the abalone Haliotis varia Linnaeus.

    PubMed

    Suja, C P; Dharmaraj, S

    2005-02-01

    The study is aimed at developing a technology for the production of in vitro pearl through tissue culture of mantle of the abalone, Haliotis varia Linnaeus, as the production of free and spherical pearls in vivo is rather difficult in abalones. In the basic study, the cell yield was intensified from the explant after 24h incubation. Among the cells liberated, the granulocytes were dominant over hyalinocytes. The size of granulocytes ranged from 3 to 16 microm and of hyalinocytes from 13 to 18 microm. Fibroblast-like cells appeared in cultures after day 2. Both granulocytes and hyalinocytes developed pseudopodial-like extensions in all directions and formed organic matrix. Granulocytes contained granules in the cytoplasm. Specific granules were responsible for nucleation of crystals. Some crystals exhibited green colour resembling mother of pearl of abalone. scanning electron microscope (SEM) study revealed the oolitic amorphous state and rhombohedral state of crystals. Its analysis through energy dispersive X-ray microanalyzer (EDS) indicated the presence of calcium. The rhombohedral crystals under polarized light showed its high birefringence (0.18) and uniaxial optically negative calcite nature with high content of calcium. A mean survival of cells was found to be 102 days in T 25 flasks and 32 days in petri dishes. Growth of cells was studied. Thirty percent of cultures were found to have contaminated during the study. The study provides basic knowledge in the development of a technology for in vitro pearl production. PMID:15695171

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

    PubMed

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

    2012-01-01

    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. PMID:21501697

  15. Insect Phylogenomics

    PubMed Central

    Behura, Susanta K.

    2015-01-01

    With the advent of next-generation sequencing methods, phylogenetics has taken a new turn in the recent years. Phylogenomics, the integration of phylogenetics with genome data, has emerged as a powerful approach to study systematics and evolution of species. Recently, breakthrough researches employing phylogenomic tools have provided better insights into the timing and pattern of insect evolution. The next-generation sequencing methods are now increasingly used by entomologists to generate genomic and transcript sequences of various insect species and strains. These data provide opportunities for comparative genomics and large-scale multigene phylogenies of diverse lineages of insects. Phylogenomic investigations help us better understand systematic and evolutionary relationships of insect species that play important roles as herbivores, predators, detritivores, pollinators, or disease vectors. It is important that we critically assess the prospects and limitations of phylogenomic methods. In this review, I describe the current status, outline the major challenges, and remark on potential future applications of phylogenomic tools in studying insect systematics and evolution. PMID:25963452

  16. The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus.

    PubMed

    Zhou, Jingxiang; Wang, Hao; Zhu, Xia; Li, Xingwei; Lv, Wenliang; Zhang, Dongming

    2013-10-01

    The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types. PMID:23893087

  17. Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

    PubMed Central

    Levi-Schaffer, F; Segal, V; Shalit, M

    1991-01-01

    We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release. PMID:2016117

  18. Temporal changes in charge content of cultured chondrocytes from bovine cartilaginous tissues.

    PubMed

    Van Damme, M P; Sinnaya, P; Derry, K; Murphy, W H; Preston, B N

    1997-03-01

    The effective charge content of the pericellular matrix of chondrocytes has been determined while the matrix is being synthesized by cells grown in culture for several weeks. The data were compared with estimates determined by chemical analysis. When measurements were performed after digestion of the matrix with papain, there was close agreement between results obtained from both techniques for proteoglycans synthesized by chondrocytes from nasal septum (a non-articular cartilage). By contrast, no such agreement was observed for proteoglycans synthesized by chondrocytes from articular cartilage, even after solubilization of the matrix with papain. While the charge calculated from chemical analysis showed a constant increase with time in culture, that measured by colloid titration showed a cyclical pattern, with maximal values occurring on days 7 and 24 of culture and a minimal value on day 14. This inability to detect all negative groups present in the matrix synthesized by articular chondrocytes would suggest the involvement of these groups in electrostatic interactions. Partial characterization of proteins synthesized by the pericellular matrix indicates that the decrease in charge content observed on day 14 could not be attributed to proteins of a particular molecular mass but possibly to an increase in the total amount of protein present. It is concluded that the marked difference in the availability of negative groups between chondrocytes cultured from articular and non-articular cartilages may reflect differences in the interaction of these negative groups with matrix components; these differences would lead to the distinct structural organization of these two cartilaginous tissues which possess different mechanical functions. PMID:9106160

  19. Ectopic Osteogenesis of Macroscopic Tissue Constructs Assembled from Human Mesenchymal Stem Cell-Laden Microcarriers through In Vitro Perfusion Culture

    PubMed Central

    Chen, Maiqin; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

    2014-01-01

    We had previously demonstrated the feasibility of preparing a centimeter-sized bone tissue construct by following a modular approach. In the present study, the objectives were to evaluate osteogenesis and tissue formation of human amniotic mesenchymal stem cells-laden CultiSpher S microcarriers during in vitro perfusion culture and after subcutaneous implantation. Microtissues were prepared in dynamic culture using spinner flasks in 28 days. In comparison with 1-week perfusion culture, microtissues became more obviously fused, demonstrating significantly higher cellularity, metabolic activity, ALP activity and calcium content while maintaining cell viability after 2-week perfusion. After subcutaneous implantation in nude mice for 6 and 12 weeks, all explants showed tight contexture, suggesting profound tissue remodeling in vivo. In addition, 12-week implantation resulted in slightly better tissue properties. However, in vitro perfusion culture time exerted great influence on the properties of corresponding explants. Degradation of microcarriers was more pronounced in the explants of 2-week perfused macrotissues compared to those of 1-week perfusion and directly implanted microtissues. Moreover, more blood vessel infiltration and bone matrix deposition with homogeneous spatial distribution were found in the explants of 2-week perfused macrotissues. Taken together, in vitro perfusion culture time is critical in engineering bone tissue replacements using such a modular approach, which holds great promise for bone regeneration. PMID:25275528

  20. Comparison of In Vitro- and Chorioallantoic Membrane (CAM)-Culture Systems for Cryopreserved Medulla-Contained Human Ovarian Tissue

    PubMed Central

    Isachenko, Vladimir; Mallmann, Peter; Petrunkina, Anna M.; Rahimi, Gohar; Nawroth, Frank; Hancke, Katharina; Felberbaum, Ricardo; Genze, Felicitas; Damjanoski, Ilija; Isachenko, Evgenia

    2012-01-01

    At present, there are three ways to determine effectively the quality of the cryopreservation procedure using ovarian tissue before the re-implantation treatment: evaluation of follicles after post-thawing xenotransplantation to SCID mouse, in-vitro culture in a large volume of culture medium under constant agitation and culture on embryonic chorio-allantoic membrane within a hen's eggs. The aim of this study was to compare the two methods, culture in vitro and culture on embryonic chorioallantoic membrane (CAM) of cryopreserved human ovarian medulla-contained and medulla-free cortex. Ovarian fragments were divided into small pieces (1.5–2.0×1.0–1.2×0.8–1.5) of two types, cortex with medulla and medulla-free cortex, frozen, thawed and randomly divided into the following four groups. Group 1: medulla-free cortex cultured in vitro for 8 days in large volume of medium with mechanical agitation, Group 2: medulla-containing cortex cultured in vitro, Group 3: medulla-free cortex cultured in CAM-system for 5 days, Group 4: medulla-containing cortex cultured in CAM-system. The efficacy of the tissue culture was evaluated by the development of follicles and by intensiveness of angiogenesis in the tissue (von Willebrand factor and Desmin). For Group 1, 2, 3 and 4, respectively 85%, 85%, 87% and 84% of the follicles were morphologically normal (P>0.1). The immunohistochemical analysis showed that angiogenesis detected by von Willebrand factor was lower in groups 1 and 3 (medulla-free cortex). Neo-vascularisation (by Desmin) was observed only in ovarian tissue of Group 4 (medulla-contained cortex after CAM-culture). It appears that the presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue. For medical practice it is recommended for evaluation of post-warming ovarian tissue to use the CAM-system as a valuable alternative to xenotransplantation and for cryopreservation of these tissues to prepare ovarian

  1. Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells.

    PubMed Central

    Kielty, C M; Boot-Handford, R P; Ayad, S; Shuttleworth, C A; Grant, M E

    1990-01-01

    The chain composition and relative abundance of type VI collagen synthesized by cells cultured from foetal bovine nuchal ligament and skin were compared with those of the type VI collagen present in these foetal tissues. Immunoprecipitation of intact collagen VI from medium and cell layers of nuchal ligament fibroblasts and skin fibroblasts at confluence revealed collagen type VI molecules with a chain composition consistent with an [alpha 1(VI)alpha 2(VI)alpha 3(VI)] monomeric assembly. Maintenance of cells in a post-confluent quiescent state promoted a marked phenotypic change in these ratios, with increased concentrations of assemblies composed of equimolar ratios of alpha 1(VI) and alpha 2(VI) chains detected in the medium of these cultures. Analysis of steady-state concentrations of mRNA for alpha 1(VI) and alpha 2(VI) chains revealed these species to be present in increased abundance at post-confluence in all the cultures, but no corresponding increase was observed in the alpha 3(VI) mRNA. In order to assess the physiological significance of these observations, the chain composition of the collagen VI content of the corresponding foetal tissues was assessed by Western blotting after extraction in guanidinium isothiocyanate under reducing conditions. Extracts of nuchal ligament revealed a collagen VI chain composition consistent with a heterotrimeric chain assembly. In contrast, the skin extracts revealed an abundance of alpha 1(VI) and alpha 2(VI) chains with only traces of the alpha 3(VI) chain detected. Increased equimolar concentrations of the alpha 1(VI)-chain and alpha 2(VI)-chain mRNAs in skin again reflected the increased concentrations of these polypeptide chains. Type VI collagen was present in greater abundance both in the nuchal ligament and in the corresponding nuchal-ligament fibroblast cultures. The results indicate that the chain composition of type VI collagen is subject to modulation at the level of transcription as a result of variations in

  2. Environmental RNAi in herbivorous insects

    PubMed Central

    Ivashuta, Sergey; Zhang, Yuanji; Wiggins, B. Elizabeth; Ramaseshadri, Partha; Segers, Gerrit C.; Johnson, Steven; Meyer, Steve E.; Kerstetter, Randy A.; McNulty, Brian C.; Bolognesi, Renata; Heck, Gregory R.

    2015-01-01

    Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism. PMID:25802407

  3. The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures

    PubMed Central

    Yen, Edwin H. K.; Sodek, Jaro; Melcher, Antony H.

    1979-01-01

    Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO2 (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [3H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO2 [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [3H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO2 [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO2 for the first 48h were unable to respond to a subsequent increase in pO2 during the last 24h. Analysis of pepsin-solubilized collagen α-chains labelled with [14C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO2. Type-III collagen comprised 20–30% of the radioactivity in α-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO2 was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo. PMID:454369

  4. Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.

    PubMed

    Farnsworth, Nikki L; Mead, Benjamin E; Antunez, Lorena R; Palmer, Amy E; Bryant, Stephanie J

    2014-11-01

    The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator. PMID:25128592

  5. Uptake, accumulation, and egress of erythromycin by tissue culture cells of human origin.

    PubMed Central

    Martin, J R; Johnson, P; Miller, M F

    1985-01-01

    The ability of erythromycin A base to penetrate and accumulate in tissue culture cells of human origin was investigated. The antibiotic was highly concentrated by early passage cells of normal bronchus, kidney, liver, lung, and skin and by cancer cells derived from breast, liver, and lung. Intracellular levels 4 to 12 times that of the extracellular milieu were obtained in both early-passage and transformed cells. The total quantity of erythromycin accumulated depended on the extracellular concentration of antibiotic, but the cellular/extracellular ratios were, for the most part, independent of the initial extracellular drug concentration. In all cell types tested, the accumulated antibiotic rapidly egressed when cells were incubated in antibiotic-free medium. Bioactivity assays demonstrated that the expelled drug was unmetabolized, fully active antibiotic. The concentration of erythromycin by a variety of human cell types probably accounts, in part, for the effectiveness of the antibiotic against intracellular parasites such as Legionella and Chlamydia spp. PMID:3994346

  6. Isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses.

    PubMed

    Schmitt, Sabine; Eberhagen, Carola; Weber, Susanne; Aichler, Michaela; Zischka, Hans

    2015-01-01

    We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66-74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66-74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities. PMID:25820716

  7. Cloning crops in a CELSS via tissue culture: Prospects and problems

    NASA Technical Reports Server (NTRS)

    Carman, John G.; Hess, J. Richard

    1990-01-01

    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.

  8. 3D Tissue Culturing: Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations (Adv. Healthcare Mater. 13/2016).

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented by M. Hagiwara and co-workers on page 1566. 3D recognition of a sample structure can be achieved by facilitating multi-directional views using a standard microscope without a laser system. The cubic platform has the potential to promote 3D culture studies, offering easy handling and compatibility with commercial culture plates at a low price tag. PMID:27384934

  9. Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

    PubMed Central

    Chiquet-Ehrismann, R; Matsuoka, Y; Hofer, U; Spring, J; Bernasconi, C; Chiquet, M

    1991-01-01

    In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices. Images PMID:1725601

  10. Curved and folded micropatterns in 3D cell culture and tissue engineering.

    PubMed

    Yilmaz, Cem Onat; Xu, Zinnia S; Gracias, David H

    2014-01-01

    Cells live in a highly curved and folded micropatterned environment within the human body. Hence, there is a need to develop engineering paradigms to replicate these microenvironments in order to investigate the behavior of cells in vitro, as well as to develop bioartificial organs for tissue engineering and regenerative medicine. In this chapter, we first motivate the need for such micropatterns based on anatomical considerations and then survey methods that can be utilized to generate curved and folded micropatterns of relevance to 3D cell culture and tissue engineering. The methods surveyed can broadly be divided into two classes: top-down approaches inspired by conventional 2D microfabrication and bottom-up approaches most notably in the self-assembly of thin patterned films. These methods provide proof of concept that the high resolution, precise and reproducible patterning of cell and matrix microenvironments in anatomically relevant curved and folded geometries is possible. A specific protocol is presented to create curved and folded hydrogel micropatterns. PMID:24560507

  11. Shear and mixing effects on cells in agitated microcarrier tissue culture reactors

    NASA Technical Reports Server (NTRS)

    Cherry, Robert S.; Papoutsakis, E. Terry

    1987-01-01

    Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from: (1) direct interaction between microcarriers and turbulent eddies; (2) collisions between microcarriers in turbulent flow; and (3) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Impeller collisions occur when beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture reactors are discussed.

  12. Rapid detection of bacterial contamination in cell or tissue cultures based on Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Bolwien, Carsten; Sulz, Gerd; Becker, Sebastian; Thielecke, Hagen; Mertsching, Heike; Koch, Steffen

    2008-02-01

    Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the critical particles are automatically baseline-corrected without using background data and classified via hierarchical cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.

  13. Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle.

    PubMed

    Lee, Denis; Norby, Kathryn; Hayes, Mitchell; Chiu, Ya-Fang; Sugden, Bill; Lambert, Paul F

    2016-01-01

    Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc. PMID:27153383

  14. Development of Soft Nanocomposite Materials and Their Applications in Cell Culture and Tissue Engineering

    PubMed Central

    Haraguchi, K

    2012-01-01

    Novel soft nanocomposite materials with unique organic/inorganic network structures have been developed by extending the strategy of “organic/inorganic nanocomposites” to the field of soft materials. The structures described here were synthesized by in-situ free-radical polymerization of various monomers in the presence of exfoliated clay (hectorite) in aqueous media. The nanocomposite hydrogels (NC gels) and soft nanocomposites (M-NCs) obtained were flexible and transparent soft materials, regardless of the clay content, that could be prepared in various shapes and surface forms, each consisting of individually different polymer/clay network structures. Owing to these unique network structures, both NC gels and M-NCs showed extraordinary mechanical properties such as ultrahigh elongation at break and widely controlled modulus and strength, which could overcome the problems (e.g., mechanical fragility, optical turbidity, poor processing ability) associated with conventional chemically crosslinked materials. In addition, the NC gels and M-NCs exhibited a number of new characteristics related to optical anisotropy, morphology, biocompatibility, stimulus sensitivity and cell culture. In the present review, we outline the novel features of these soft nanocomposites, and demonstrate their potential as soft culture substrates useful for tissue engineering as well as soft, transparent, absorbing, and mechanically tough biomaterials for many bio-applications. PMID:24693187

  15. Achondrogenesis type IB (Fraccaro): study of collagen in the tissue and in chondrocytes cultured in agarose.

    PubMed

    Freisinger, P; Stanescu, V; Jacob, B; Cohen-Solal, L; Maroteaux, P; Bonaventure, J

    1994-02-15

    A lethal chondrodysplasia characterized by extreme micromelia was diagnosed by ultrasound examination in two sibs whose nonconsanguineous parents were healthy. Radiographic and histopathologic data indicated that the two foetuses (18 and 21 weeks old) had achondrogenesis type IB (Fraccaro). Quantitation of total collagen extractable from dried cartilage samples demonstrated a 50% decrease when compared to an age-related control. This decrease was essentially related to type II collagen. Nevertheless, the alpha chains and the CB peptides of type II collagen had a normal electrophoretic mobility. A significant amount of collagen type I was also detected. The electrophoretic pattern of collagens type IX and XI did not differ significantly from control sample. The extracellular matrix elaborated by patient chondrocytes cultured in agarose for 10-12 days, contained less collagen type II than normal cells. Labelling with 14C-proline of cultured cells showed the presence of procollagen and type II collagen chains with a normal electrophoretic mobility, but an alpha 2(I) chain was detectable in the patient material, indicating the presence of collagen type I which supported the tissue findings. The significance of the type II collagen reduction in the patient's cartilage is unclear but it is unlikely to be the primary defect in achondrogenesis type I. PMID:8160740

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

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    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-aminopyridine (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 receptor-associated protein (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. PMID:26500501

  17. Differences in protein binding and cytokine release from monocytes on commercially sourced tissue culture polystyrene.

    PubMed

    Battiston, Kyle G; McBane, Joanne E; Labow, Rosalind S; Paul Santerre, J

    2012-01-01

    Tissue culture polystyrene (TCPS) is a ubiquitous substrate used by many researchers in the biomedical and biological sciences. Different parameters involved in the production of TCPS, including the treatment time and the use of reactive gases and chemical agents, can have a significant influence on the ultimate surface properties achieved. The assumption that they will all yield a consistent and controlled product has not proven to be true. To provide a better insight into the bioactivity differences in TCPS supplied by different manufacturers, TCPS from three different companies (Sarstedt, Wisent Corp., and Becton Dickinson (BD)) were analyzed for their surface properties, protein adsorption characteristics, and interactions with human monocytes. Marked differences were observed in terms of surface wettability and surface chemistry. Furthermore, Wisent TCPS adsorbed more than twice the amount of serum proteins compared with BD and Sarstedt TCPS. Sarstedt showed significantly more cell retention (more DNA) compared with both BD and Wisent TCPS brands over a 7 day culture period. Cytokine release from monocytes adherent on the three different TCPS also differed significantly, suggesting that the differences in the surface properties were sufficient to differentially mediate monocyte activation. These results have important implications for TCPS research use, in terms of appreciating the interpretation of the data when TCPS is used as a control substrate as well as when it is used where a pre-conditioned state would influence the outcome of the study. PMID:21963405

  18. A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion.

    PubMed

    Kenar, Halime; Kose, Gamze T; Toner, Mehmet; Kaplan, David L; Hasirci, Vasif

    2011-08-01

    The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Wharton's Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Wharton's Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure. PMID:21570112

  19. Expanding the genetic variability of flatpea using tissues culture, mutagenesis, and intercrossing techniques

    SciTech Connect

    Coulombe, B.A.

    1988-01-01

    Flatpea (Lathyrus sylvestris L.) is a potentially valuable forage legume but contains high levels of 2,4-diaminobutyric acid (DABA), a compound that can have adverse effects on some animals, including rats and poultry. To increase genetic variability in foliar DABA content and other traits of interest, three approaches were utilized: (1) regeneration of flatpea plants from tissue culture to produce potential somaclonal variants, (2) seed irradiation and screening of potentially mutated progeny, and (3) intercrossing among flatpea accessions. Low-frequency whole plant regeneration of flatpea was obtained from hypocotyl-derived callus cultures. Initial tests established that the effective range of gamma-irradiation for seed treatment was between 10.0 and 17.5 kR. Within this range, reduction in percentage of both seedling height and plant survival was a linear function of dose. Individual M{sub 2} plants that contained reduced levels of DABA were identified. No significant trend in DABA concentration with increasing gamma irradiation was apparent. Flatpea pollination methods were evaluated prior to utilization of intercrossing for inducing genetic variability. Appropriate flower stages for emasculation were determined by in vitro germination of pollen. Lines that produced high numbers of seeds per pollination were identified by crossing in all possible combinations among seven flatpea accessions.

  20. Immunofluorescence detection of the cytoskeleton and extracellular matrix in tissue and cultured cells.

    PubMed

    Smith-Clerc, Josiane; Hinz, Boris

    2010-01-01

    "A picture is worth a thousand words" goes the proverb. A poor picture however can be worse than saying nothing at all. This is particularly true for immunofluorescence pictures that in addition to their informative character bear an esthetic component. We here provide a panel of straightforward methods to process tissue sections and cultured cells for immunostaining of cytoskeletal elements, primarily those associated with actin filaments. We want to emphasize to the reader the fact that the choice of the processing method will have an important influence on the outcome of the immunostaining and thus on the interpretation of the results. Fixation of cultured cells with cross-linking reagents such as paraformaldehyde efficiently preserves structural elements at the expense of reduced antigenicity. The degree and timing of cell permeabilization with detergents, along with chemical cross-linking, contributes to the clarity and resolution of distinct structures but can also lead to loss of information. Fixation with organic solvents like methanol will, in most cases, better preserve antigens but will produce a higher background and impact on structural integrity. Therefore, it is recommended to test different protocols for a "new" protein or epitope - the results will pay back your investment. PMID:19960321

  1. Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    PubMed Central

    Abdul-Hussein, Saba; Rahl, Karin; Moslemi, Ali-Reza; Tajsharghi, Homa

    2013-01-01

    Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein. PMID:24039757

  2. Biological effect of radiation-degraded alginate on flower plants in tissue culture.

    PubMed

    Le, Q Luan; Nguyen, Q Hien; Nagasawa, Naotsugu; Kume, Tamikazu; Yoshii, Fumio; Nakanishi, Tomoko M

    2003-12-01

    Alginate with a weight-average molecular mass (Mw) of approx. 9.04 x 10(5) Da was irradiated at 10-200 kGy in 4% (w/v) aqueous solution. The degraded alginate product was used to study its effectiveness as a growth promoter for plants in tissue culture. Alginate irradiated at 75 kGy with an Mw of approx. 1.43 x 10(4) Da had the highest positive effect in the growth of flower plants, namely limonium, lisianthus and chrysanthemum. Treatment of plants with irradiated alginate at concentrations of 30-200 mg/l increased the shoot multiplication rate from 17.5 to 40.5% compared with control. In plantlet culture, 100 mg/l irradiated alginate supplementation enhanced shoot height (9.7-23.2%), root length (9.7-39.4%) and fresh biomass (8.1-19.4%) of chrysanthemum, lisianthus and limonium compared with that of the untreated control. The survival ratios of the transferred flower plantlets treated with irradiated alginate were almost the same as the control value under greenhouse conditions. However, better growth was attained for the treated plantlets. PMID:12901723

  3. Constructing Failure: Leonard Hayflick, Biomedicine, and the Problems with Tissue Culture.

    PubMed

    Park, Hyung Wook

    2016-07-01

    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. PMID:26236962

  4. Expression of Alcohol Dehydrogenase 3 in Tissue and Cultured Cells from Human Oral Mucosa

    PubMed Central

    Hedberg, Jesper J.; Höög, Jan-Olov; Nilsson, Jan A.; Xi, Zheng; Elfwing, Åsa; Grafström, Roland C.

    2000-01-01

    Because formaldehyde exposure has been shown to induce pathological changes in human oral mucosa, eg, micronuclei, the potential enzymatic defense by alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase was characterized in oral tissue specimens and cell lines using RNA hybridization and immunological methods as well as enzyme activity measurements. ADH3 mRNA was expressed in basal and parabasal cell layers of oral epithelium, whereas the protein was detected throughout the cell layers. ADH3 mRNA and protein were further detected in homogenates of oral tissue and various oral cell cultures, including, normal, SV40T antigen-immortalized, and tumor keratinocyte lines. Inhibition of the growth of normal keratinocytes by maintenance at confluency significantly decreased the amount of ADH3 mRNA, a transcript with a determined half-life of 7 hours. In contrast, decay of ADH3 protein was not observed throughout a 4-day period in normal keratinocytes. In samples from both tissue and cells, the ADH3 protein content correlated to oxidizing activity for the ADH3-specific substrate S-hydroxymethylglutathione. The composite analyses associates ADH3 mRNA primarily to proliferative keratinocytes where it exhibits a comparatively short half-life. In contrast, the ADH3 protein is extremely stable, and consequently is retained during the keratinocyte life span in oral mucosa. Finally, substantial capacity for formaldehyde detoxification is shown from quantitative assessments of alcohol- and aldehyde-oxidizing activities including Km determinations, indicating that ADH3 is the major enzyme involved in formaldehyde oxidation in oral mucosa. PMID:11073833

  5. Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

    PubMed

    Uhl, Franziska E; Vierkotten, Sarah; Wagner, Darcy E; Burgstaller, Gerald; Costa, Rita; Koch, Ina; Lindner, Michael; Meiners, Silke; Eickelberg, Oliver; Königshoff, Melanie

    2015-10-01

    Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs. PMID:25929950

  6. Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate.

    PubMed

    Maund, Sophia Lisette; Nolley, Rosalie; Peehl, Donna Mae

    2014-02-01

    Few preclinical models accurately depict normal human prostate tissue or primary prostate cancer (PCa). In vitro systems typically lack complex cellular interactions among structured prostatic epithelia and a stromal microenvironment, and genetic and molecular fidelity are concerns in both in vitro and in vivo models. 'Tissue slice cultures' (TSCs) provide realistic preclinical models of diverse tissues and organs, but have not been fully developed or widely utilized for prostate studies. Problems encountered include degeneration of differentiated secretory cells, basal cell hyperplasia, and poor survival of PCa. Here, we optimized, characterized, and applied a TSC model of primary human PCa and benign prostate tissue that overcomes many deficiencies of current in vitro models. Tissue cores from fresh prostatectomy specimens were precision-cut at 300 μm and incubated in a rotary culture apparatus. The ability of varied culture conditions to faithfully maintain benign and cancer cell and tissue structure and function over time was evaluated by immunohistological and biochemical assays. After optimization of the culture system, molecular and cellular responses to androgen ablation and to piperlongumine (PL), purported to specifically reduce androgen signaling in PCa, were investigated. Optimized culture conditions successfully maintained the structural and functional fidelity of both benign and PCa TSCs for 5 days. TSCs exhibited androgen dependence, appropriately undergoing ductal degeneration, reduced proliferation, and decreased prostate-specific antigen expression upon androgen ablation. Further, TSCs revealed cancer-specific reduction of androgen receptor and increased apoptosis upon treatment with PL, validating data from cell lines. We demonstrate a TSC model that authentically recapitulates the structural, cellular, and genetic characteristics of the benign and malignant human prostate, androgen dependence of the native tissue, and cancer-specific response

  7. Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage.

    PubMed

    Kang, Ning; Liu, Xia; Guan, Yue; Wang, Jian; Gong, Fuxing; Yang, Xun; Yan, Li; Wang, Qian; Fu, Xin; Cao, Yilin; Xiao, Ran

    2012-06-01

    Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1. PMID:22440049

  8. Selective enhancement of scopadulcic acid B production in the cultured tissues of Scoparia dulcis by methyl jasmonate.

    PubMed

    Nkembo, Kasidimoko Marguerite; Lee, Jung-Bum; Hayashi, Toshimitsu

    2005-07-01

    The effects of methyl jasmonate (MeJA) on isoprenoid production were evaluated in cultured tissues of Scoparia dulcis. It was found that MeJA suppressed the accumulation of chlorophylls, carotenoids, phytol and beta-sitosterol in the tissues. MeJA, however, remarkably enhanced the production of scopadulcic acid B (SDB), with 10 microM being optimal observed concentration for stimulation of SDB production. The maximum concentration of SDB was observed 6 d after MeJA treatment. PMID:15997134

  9. Insects: A nutritional alternative

    NASA Technical Reports Server (NTRS)

    Dufour, P. A.

    1981-01-01

    Insects are considered as potential food sources in space. Types of insects consumed are discussed. Hazards of insect ingestion are considered. Insect reproduction, requirements, and raw materials conversion are discussed. Nutrition properties and composition of insects are considered. Preparation of insects as human food is discussed.

  10. Lysosome biogenesis/scattering increases host cell susceptibility to invasion by Trypanosoma cruzi metacyclic forms and resistance to tissue culture trypomastigotes.

    PubMed

    Cortez, Cristian; Real, Fernando; Yoshida, Nobuko

    2016-05-01

    A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect-borne and mammalian-stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient-deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose-induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR-associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT-specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome-dependent, whereas TCT entry is predominantly lysosome-independent. PMID:26572924

  11. Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: a novel approach to reduce oxidative browning in plant tissue culture.

    PubMed

    Jones, Andrew Maxwell Phineas; Saxena, Praveen Kumar

    2013-01-01

    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. PMID:24116165

  12. Inhibition of Phenylpropanoid Biosynthesis in Artemisia annua L.: A Novel Approach to Reduce Oxidative Browning in Plant Tissue Culture

    PubMed Central

    Jones, Andrew Maxwell Phineas; Saxena, Praveen Kumar

    2013-01-01

    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. PMID:24116165

  13. Insect evolution.

    PubMed

    Engel, Michael S

    2015-10-01

    It goes without saying that insects epitomize diversity, and with over a million documented species they stand out as one of the most remarkable lineages in the 3.5-billion-year history of life on earth (Figure 1). This reality is passé to even the layperson and is taken for granted in the same way none of us think much of our breathing as we go about our day, and yet insects are just as vital to our existence. Insects are simultaneously familiar and foreign to us, and while a small fraction are beloved or reviled, most are simply ignored. These inexorable evolutionary overachievers outnumber us all, their segmented body plan is remarkably labile, they combine a capacity for high rates of speciation with low levels of natural extinction, and their history of successes eclipses those of the more familiar ages of dinosaurs and mammals alike. It is their evolution - persisting over vast expanses of geological time and inextricably implicated in the diversification of other lineages - that stands as one of the most expansive subjects in biology. PMID:26439349

  14. Investigation of the in vitro culture process for skeletal-tissue-engineered constructs using computational fluid dynamics and experimental methods.

    PubMed

    Hossain, Md Shakhawath; Chen, X B; Bergstrom, D J

    2012-12-01

    The in vitro culture process via bioreactors is critical to create tissue-engineered constructs (TECs) to repair or replace the damaged tissues/organs in various engineered applications. In the past, the TEC culture process was typically treated as a black box and performed on the basis of trial and error. Recently, computational fluid dynamics (CFD) has demonstrated its potential to analyze the fluid flow inside and around the TECs, therefore, being able to provide insight into the culture process, such as information on the velocity field and shear stress distribution that can significantly affect such cellular activities as cell viability and proliferation during the culture process. This paper briefly reviews the CFD and experimental methods used to investigate the in vitro culture process of skeletal-type TECs in bioreactors, where mechanical deformation of the TEC can be ignored. Specifically, this paper presents CFD modeling approaches for the analysis of the velocity and shear stress fields, mass transfer, and cell growth during the culture process and also describes various particle image velocimetry (PIV) based experimental methods to measure the velocity and shear stress in the in vitro culture process. Some key issues and challenges are also identified and discussed along with recommendations for future research. PMID:23363205

  15. FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

    PubMed

    Aguiar, F L N; Lunardi, F O; Lima, L F; Rocha, R M P; Bruno, J B; Magalhães-Padilha, D M; Cibin, F W S; Nunes-Pinheiro, D C S; Gastal, M O; Rodrigues, A P R; Apgar, G A; Gastal, E L; Figueiredo, J R

    2016-04-01

    This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture. PMID:26723132

  16. Characteristics of trophoblastic tissue derived from in vitro culture of preimplantation embryos of the common marmoset monkey.

    PubMed

    Summers, P M; Taylor, C T; Hearn, J P

    1987-01-01

    The features of trophoblastic tissue derived from the in vitro culture of marmoset monkey embryos have been described. Long-term trophoblast cultures (in excess of three years in one case) were established from the primary trophoblast monolayer of four of 38 embryos; division of one of these embryos produced two long-term cultures. The trophoblast cells retained their ability to synthesize and secrete chorionic gonadotrophin (CG) during maintenance in vitro and were capable of prolonging the luteal phase when transferred to the uterus of marmosets. A characteristic feature of the cultures was the formation of multiple fluid-filled vesicles enclosed by a single layer of cytotrophoblast cells and attached to the culture dish by a small monolayer of syncytiotrophoblast cells. The tissue was propagated by cutting vesicles into small pieces and placing into a fresh culture dish; attempts to subculture using single-cell suspensions were unsuccessful. These cultures provide a convenient source of marmoset CG for purification as well as an in vitro system for studying other secretory products of primate trophoblast. PMID:3120174

  17. DNA Methylation and Histone Acetylation Patterns in Cultured Bovine Adipose Tissue-Derived Stem Cells (BADSCs)

    PubMed Central

    Abouhamzeh, Beheshteh; Salehi, Mohammad; Hosseini, Ahmad; Masteri-Farahani, Ali Reza; Fadai, Fatemeh; Heidari, Mohammad Hasan; Nourozian, Mohsen; Soleimani, Masoud; Khorashadizadeh, Mohsen; Mossahebi-Mohammadi, Majid; Mansouri, Ardalan

    2015-01-01

    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. PMID:25685737

  18. Genomic sequence analysis of the United States infectious laryngotracheitis vaccine strains chicken embryo origin (CEO) and tissue culture origin (TCO)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomic sequences of low and high passages of U.S. infectious laryngotracheitis (ILT) vaccine strains chicken embryo origin (CEO) and tissue culture origin (TCO) these strains were determined using hybrid next generation sequencing in order to define relevant genomic changes associated with att...

  19. Candidate Genes Within Tissue Culture Regeneration QTL Revisited with a Linkage Map Based on Transcript Derived Markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Green plant regeneration from tissue culture is under the genetic control of multiple genes. Candidate genes for regeneration have been identified in multiple species using QTL and microarray analyses, and some of these genes have been verified as improving regeneration through transformation. Multi...

  20. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    PubMed Central

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    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 of appropriate

  1. Tissue culture and explant approaches to studying and visualizing Neospora caninum and its interactions with the host cell.

    PubMed

    Hemphill, Andrew; Vonlaufen, Nathalie; Naguleswaran, Arunasalam; Keller, Nadine; Riesen, Michele; Guetg, Nicole; Srinivasan, Sangeetha; Alaeddine, Ferial

    2004-10-01

    Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells. PMID:15525434

  2. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

    PubMed

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

    2015-10-01

    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. PMID:26337939

  3. Insect abatement system

    NASA Technical Reports Server (NTRS)

    Spiro, Clifford Lawrence (Inventor); Burnell, Timothy Brydon (Inventor); Wengrovius, Jeffrey Hayward (Inventor)

    1997-01-01

    An insect abatement system prevents adhesion of insect debris to surfaces which must be kept substantially free of insect debris. An article is coated with an insect abatement coating comprising polyorganosiloxane with a Shore A hardness of less than 50 and a tensile strength of less than 4 MPa. A method for preventing the adhesion of insect debris to surfaces includes the step of applying an insect abatement coating to a surface which must be kept substantially free of insect debris.

  4. Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Nai-dong; Chen, Han; Li, Jun; Sang, Mang-mang; Ding, Shen; Yu, Hao

    2015-04-01

    The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800-600 cm-1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

  5. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

    PubMed

    Knight, Eleanor; Przyborski, Stefan

    2015-12-01

    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. PMID:25411113

  6. Prevalence, site and tissue preference of myxozoan parasites infecting gills of cultured fish in Punjab (India).

    PubMed

    Kaur, Harpreet; Katoch, Anu

    2016-02-25

    Native carp species cultured in Indian farms in Punjab (catla Catla catla, rohu Labeo rohita, mrigal Cirrhinus mrigala, exotic carps such as silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella, common carp Cyprinus carpio and a catfish Sperata seenghala) were examined for the presence of myxozoan parasites infecting gills. Firstly, the gills were examined under a zoom-stereomicroscope for the presence of plasmodia. The number of plasmodia per gill was counted to determine the index for the intensity of infection. Infected tissues were processed for histology, and 3-4 µm sections of infected gills were stained with haematoxylin & eosin and Luna's method. A total of 19 species of myxosporean were found infecting various cell types in the gills. Of these, 14 species belonged to the genus Myxobolus, 3 species to the genus Thelohanellus and 2 species to the genus Henneguya. Species belonging to the genus Myxobolus formed the interlamellar and intralamellar vascular (LV) type plasmodia, and species belonging to the genus Thelohanellus and Henneguya formed intrafilamental vascular (FV) type plasmodia. Mixed infections comprising 2, 3 or 4 different myxozoan species were noted in individual fish. The most common type of parasitism was polyparasitism due to 4 myxobolids co-occuring in fish with an infection rate of 23.16%. All species caused mild to severe haemorrhagic gill disease with little clinical symptomatology. PMID:26912043

  7. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

    PubMed Central

    Baghirova, Sabina; Hughes, Bryan G.; Hendzel, Michael J.; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C PMID:26740924

  8. Controlled surface modification of tissue culture polystyrene for selective cell binding using resilin-inspired polypeptides.

    PubMed

    Vashi, Aditya V; Ramshaw, John A M; Glattauer, Veronica; Elvin, Christopher M; Lyons, Russell E; Werkmeister, Jerome A

    2013-09-01

    Modified tissue culture polystyrene (TCP) surfaces have been fabricated by attachment of recombinant polypeptides based on Drosophila melanogaster resilin and the Anopheles gambiae resilin-like protein. The D. melanogaster polypeptide (Rec-1) was from the first exon of resilin and consisted of 17 very similar repeats of a 15 residue sequence. The A. gambiae polypeptide consisted of 16 repeats of an 11 residue consensus sequence (An16). Polypeptides were attached to the TCP surface through tyrosine-based photo-crosslinking using blue light in combination with (RuII(bpy)3)Cl2 and sodium persulfate. TCP that has been manufactured by mild oxidation has surface phenolic groups that are believed to participate in this crosslinking process. X-ray photoelectron spectroscopy and contact angle analyses were used to demonstrate polypeptide binding. At higher coating concentrations of Rec-1 and An16, the surface was passivated and fibroblasts no longer attached and spread. At coating concentrations of 1 mg ml(-1) for Rec-1 and 0.1 mg ml(-1) for An16, where the surface was fully passivated against fibroblast attachment, addition of a cell attachment peptide, cyclo(Arg-Gly-Asp-D-Tyr-Lys) during coating and photo-crosslinking at >0.1 mg ml(-1), led to the restoration of fibroblast binding that was dependent on the integrin αV chain. PMID:23748293

  9. Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis.

    PubMed Central

    Jura, H; Bader, A; Hartmann, M; Maschek, H; Frosch, M

    1996-01-01

    An in vitro model for growth and differentiation of the metacestode tissue of the tapeworm Echinococcus multilocularis is described. This model simulates the organotropism of the parasite toward the liver of the intermediate host. In the presence of collagen-embedded primary hepatocytes from rats and humans, which can be kept in culture for 2 to 3 months, the parasitic vesicles grew by exogenous budding and multiplied about 12-fold within 3 weeks. In contrast, without the hepatocytes, the metacestodes rapidly degenerated. Development of protoscolices was seen only in the presence of rat hepatocytes but not in coculture of the metacestodes with hepatocytes of human origin, thus reflecting the in vivo situation during infection of rodents and in alveolar echinococcosis in humans. The experiments indicated that growth of the metacestodes and development of protoscolices depended on soluble low-molecular-weight factors released by the hepatocytes. The in vitro-grown metacestodes did not differ morphologically from the larvae found in infected intermediate hosts, and their infectivity was completely maintained. This report describes the first in vitro model of alveolar echinococcosis and will be the basis for future studies on host-parasite interactions of this important zoonosis. PMID:8751888

  10. Three-dimensional Micro-culture System for Tooth Tissue Engineering.

    PubMed

    Kuchler-Bopp, S; Bécavin, T; Kökten, T; Weickert, J L; Keller, L; Lesot, H; Deveaux, E; Benkirane-Jessel, N

    2016-06-01

    The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development. Progress is being made to regenerate teeth by reassociating dissociated embryonic dental cells and implanting them in vivo. In the present study, we tested the hanging drop method to study mixed epithelial-mesenchymal cell reorganization in a liquid instead of semisolid medium to see whether it could lead to tooth histogenesis and organogenesis. This method allowed the control of the proportion and number of cells to be used, and the forming microtissues showed homogeneous size. The liquid environment favored cell migrations as compared with collagen gels. Three protocols were compared. The one that sequentially combined the hanging drop and semisolid medium cultures prior to in vivo implantation gave the best results. Indeed, after implantation, teeth developed, showing a well-formed crown, mineralization of dentin and enamel, and the initiation of root formation. Vascularization and the cellular heterogeneity in the mesenchyme were similar to what was observed in developing molars. Finally, after coimplantation with a trigeminal ganglion, the dental mesenchyme, including the odontoblast layer, became innervated. The real advantage of this technique is the small number of cells required to make a tooth. This experimental model can be employed to study the development, physiology, metabolism, or toxicology in forming teeth and test other cell sources. PMID:26965424

  11. Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition

    PubMed Central

    Mohammadzadeh, Fatemeh; Mosayebi, Gholamali; Montazeri, Vahid; Darabi, Maryam; Fayezi, Shabnam; Shaaker, Maghsod; Rahmati, Mohammad; Baradaran, Behzad; Mehdizadeh, Amir

    2014-01-01

    Purpose Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. Methods Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. Results Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 µM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. Conclusion The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition

  12. A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings.

    PubMed

    Laukkanen, H; Soini, H; Kontunen-Soppela, S; Hohtola, A; Viljanen, M

    2000-07-01

    We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.). The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture. Growth of Scots pine cells had ceased in all of these cultures. Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales. Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species. Its sequence most closely resembled those of Mycobacterium obuense (97%) and M. aichiense (96%). Tissue browning was frequently observed in callus or suspension culture of Scots pine. Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions. Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants. However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M. chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium. Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M. chlorophenolicum or Pseudomotnas fluorescens. We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree. Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study. PMID:11303582

  13. Genomics of Insect-Soybean Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The dissection of plant-insect interactions has lagged behind that of interactions between plants and other types of pests. Insect pests interact with plants in a variety of ways, ranging from piercing and sucking of phloem to consumption of leaves and other tissues. Hence, a wide range of genetic m...

  14. A novel bidirectional continuous perfusion bioreactor for the culture of large-sized bone tissue-engineered constructs.

    PubMed

    Gardel, Leandro S; Correia-Gomes, Carla; Serra, Luís A; Gomes, Manuela E; Reis, Rui L

    2013-11-01

    This works reports the development and preliminary assessment of a new bioreactor for culturing large-sized three-dimensional constructs in bone tissue engineering. The bidirectional continuous perfusion bioreactor (BCPB) promotes mechanical stimulation of cells through the creation of shear forces induced by flow perfusion. The main innovation consists in the possibility of culturing scaffolds of large dimensions that can be suitable for the regeneration of critical sized defects. The functionality of BCPB was preliminarily evaluated by culturing starch-polycaprolactone scaffolds/goat bone marrow stromal cells for 14 and 21 days. Cylindrical blocks were stacked (42 mm thick). Static culture was used as controls. The samples were collected for DNA, alkaline phosphatase (ALP), scanning electron microscopy (SEM), and histological analysis. The results showed higher ALP levels in the bioreactor cultures than those obtained under static conditions. The number of cells in constructs cultured in the bioreactor showed lower values compared to static cultures, suggesting that static conditions tend to privilege the metabolic path way for cellular proliferation while dynamic conditions tend to privilege the metabolic path for osteogenic differentiation. SEM observations show that, the migration and cell distribution was observed in the bioreactor. These results demonstrate the feasibility and the benefit of culturing constructs in BCPB. PMID:23681695

  15. Somaclonal Variation Is Induced De Novo via the Tissue Culture Process: A Study Quantifying Mutated Cells in Saintpaulia

    PubMed Central

    Sato, Mitsuru; Hosokawa, Munetaka; Doi, Motoaki

    2011-01-01

    Background The origin of somaclonal variation has not been questioned previously, i.e., “pre-existing mutations” in explants and “newly induced mutations” arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation. Methodology/Principal Findings We adopted a petal-variegated cultivar of Saintpaulia ‘Thamires’ (Saintpaulia sp.) as the model plant. Based on the difference between the pre- and post-transposon excision sequence of the promoter region of flavonoid 3′, 5′-hydoroxylase (F3′5′H), we estimated mutated (transposon-excised) cell percentages using a quantitative real-time PCR. Mutated cell percentages in leaf laminae used as explants was 4.6 and 2.4% in highly or low variegation flower plants, respectively, although the occurrences of blue color mutants in their regenerants were more than 40%. Preexisting mutated cell percentages in cultured explants were considerably lower than the mutated plant percentage among total regenerants via tissue culture. Conclusions/Significance The estimation of mutated cell percentages became possible using the quantitative real-time PCR. The origins of mutations were successfully distinguished; it was confirmed that somaclonal variations are mainly caused by newly generated mutations arising from tissue culture process. PMID:21853148

  16. Single nanoparticle tracking of [Formula: see text]-methyl-d-aspartate receptors in cultured and intact brain tissue.

    PubMed

    Varela, Juan A; Ferreira, Joana S; Dupuis, Julien P; Durand, Pauline; Bouchet, Delphine; Groc, Laurent

    2016-10-01

    Recent developments in single-molecule imaging have revealed many biological mechanisms, providing high spatial and temporal resolution maps of molecular events. In neurobiology, these techniques unveiled that plasma membrane neurotransmitter receptors and transporters laterally diffuse at the surface of cultured brain cells. The photostability of bright nanoprobes, such as quantum dots (QDs), has given access to neurotransmitter receptor tracking over long periods of time with a high spatial resolution. However, our knowledge has been restricted to cultured systems, i.e., neurons and organotypic slices, therefore lacking several aspects of the intact brain rheology and connectivity. Here, we used QDs to track single glutamatergic [Formula: see text]-methyl-d-aspartate receptors (NMDAR) in acute brain slices. By delivering functionalized nanoparticles in vivo through intraventricular injections to rats expressing genetically engineered-tagged NMDAR, we successfully tracked the receptors in native brain tissue. Comparing NMDAR tracking to different classical brain preparations (acute brain slices, cultured organotypic brain slices, and cultured neurons) revealed that the surface diffusion properties shared several features and are also influenced by the nature of the extracellular environment. Together, we describe the experimental procedures to track plasma membrane NMDAR in dissociated and native brain tissue, paving the way for investigations aiming at characterizing receptor diffusion biophysics in intact tissue and exploring the physiopathological roles of receptor surface dynamics. PMID:27429996

  17. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

    Avila, Frank W.; Sirot, Laura K.; LaFlamme, Brooke A.; Rubinstein, C. Dustin; Wolfner, Mariana F.

    2014-01-01

    Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects. PMID:20868282

  18. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation

    PubMed Central

    Smitz, J.; Dolmans, M.M.; Donnez, J.; Fortune, J.E.; Hovatta, O.; Jewgenow, K.; Picton, H.M.; Plancha, C.; Shea, L.D.; Stouffer, R.L.; Telfer, E.E.; Woodruff, T.K.; Zelinski, M.B.

    2010-01-01

    BACKGROUND Female cancer patients are offered ‘banking’ of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research. PMID:20124287

  19. Expression of the Peptide Antibiotic Human β-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

    PubMed Central

    Krisanaprakornkit, Suttichai; Weinberg, Aaron; Perez, Christopher N.; Dale, Beverly A.

    1998-01-01

    Human β-defensin-1 (hBD-1) is a member of the family of small cationic antimicrobial peptides that have been identified in several mucosal epithelia. Because human gingival epithelium is a site that is constantly challenged by oral microorganisms, we examined the expression of hBD-1 in human gingival epithelial and fibroblast cell cultures and tissue samples. Cell cultures were challenged with cell wall extracts of Porphyromonas gingivalis or Fusobacterium nucleatum, Escherichia coli lipopolysaccharide, tumor necrosis factor alpha, or phorbol myristate acetate. hBD-1 mRNA was detected in unstimulated and stimulated cultures by reverse transcription (RT)-PCR using several primer sets specific for hBD-1. Gingival epithelial cells, but not gingival fibroblasts, expressed a product of the predicted size for hBD-1 mRNA. The sequence of the PCR product was identical to that of hBD-1. hBD-1 mRNA expression was not significantly modulated by any of the stimulants tested. Human gingival tissues from noninflamed and inflamed sites were also analyzed by RT-PCR. hBD-1 mRNA was expressed in all tissue samples. The relative expression of hBD-1 mRNA was similar in noninflamed and inflamed tissues obtained from each of four patients undergoing treatment for periodontitis. However, the relative expression of hBD-1 mRNA varied in gingival biopsies obtained from 15 different normal individuals, and the relative hBD-1 expression was unrelated to interleukin-8 expression. Our findings show the constitutive expression of hBD-1 mRNA in cultured epithelial cells and gingival tissues but not gingival fibroblasts. These findings suggest that expression of hBD-1 may play a role as part of the innate host defenses in maintaining normal gingival health. PMID:9712771

  20. Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids

    PubMed Central

    2013-01-01

    Background Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. Results We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Conclusions Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of

  1. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle.

    PubMed Central

    Guharay, F; Sachs, F

    1984-01-01

    The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+-activated K+ channels are not affected by stretch. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of +30 mV. Kinetic analysis of single-channel records indicates that there are one open (O) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C3-O. Only the rate constant that governs the C1-C2 transition (k1,2) is stretch-sensitive. None of the rates are voltage-sensitive. The rate constant k1,2 varies with the square of the tension as k1, 2 = k0 X e alpha T2, where alpha is a constant describing the sensitivity to stretch and T is the tension. A typical value of alpha is 0.08 (dyn cm-1)-2. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, alpha, increases from 0.08 to 2.4 (dyn cm-1)-2. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0.004 in normal saline (5 mM-K+) to 0.26 in 150 mM-K+. The channel appears to gather force from a large area of membrane (greater than 3 X 10(5) A2), probably by a cytochalasin-resistant cytoskeletal network. PMID:6086918

  2. Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction.

    PubMed

    Jennewein, Martina; Bubel, Monika; Guthörl, Silke; Metzger, Wolfgang; Weigert, Martin; Pohlemann, Tim; Oberringer, Martin

    2016-08-01

    The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing. PMID:27026609

  3. Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

    PubMed Central

    Templeton, Zachary S.; Bachmann, Michael H.; Alluri, Rajiv V.; Maloney, William J.; Contag, Christopher H.; King, Bonnie L.

    2015-01-01

    Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues. PMID:25867136

  4. Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

    PubMed

    Lee, J H; Fitzgerald, J B; DiMicco, M A; Cheng, D M; Flannery, C R; Sandy, J D; Plaas, A H; Grodzinsky, A J

    2009-09-01

    We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury. PMID:19607802

  5. Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.

    PubMed

    Hagiwara, Masaya; Kawahara, Tomohiro; Nobata, Rina

    2016-07-01

    An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles. PMID:27128576

  6. Allergies to Insect Venom

    MedlinePlus

    ... The smell of food attracts these insects.  Use insect repellents and keep insecticide available. Treatment tips:  Venom immunotherapy (allergy shots to insect venom(s) is highly effective in preventing subsequent sting ...

  7. Growth of plant tissue cultures in simulated lunar soil: Implications for a lunar base Controlled Ecological Life Support System (CELSS)

    NASA Technical Reports Server (NTRS)

    Venketeswaran, S.

    1987-01-01

    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.

  8. Two-photon imaging of collagen remodeling in RAFT tissue cultures

    NASA Astrophysics Data System (ADS)

    Wallace, Vincent P.; Coleno, Mariah L.; Yomo, Tatsuro; Sun, Chung-Ho; Tromberg, Bruce J.

    2001-04-01

    Tissue remodeling is associated with both normal and abnormal processes including wound healing, fibrosis and cancer. In skin, abnormal remodeling causes permanent structural changes that can lead to hypertropic scarring and keloid formation. Normal remodeling, although fast and efficient in skin, is still imperfect, and a connective tissue scar remains at the wound site1. As a result, methods are needed to optimize tissue remodeling in vivo in all cases of wound repair. Since fibroblast-mediated contraction of engineered 3-D collagen based tissues (RAFTs) represents an in vitro model of the tissue contraction and collagen remodeling that occurs in vivo, RAFT tissue contraction studies combined with two-photon microscopy (TPM) studies are used to provide information on ways to improve tissue remodeling in vivo. In the RAFT models discussed here, tissue contraction is modulated either by application of exogenous growth factors or photodynamic therapy. During tissue contraction, TPM is used to image changes in Collagen Type I fibers in the RAFT skin models. Tissues are imaged at depth at day 15 after modulation. TPM signal analysis shows that RAFT tissues having the highest collagen density have the fastest rate of decay of fluorescent signal with depth.

  9. Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium.

    PubMed

    Eyestone, W H; First, N L

    1989-03-01

    In Exp. 1, 5-8-cell embryos from superovulated cattle were co-cultured with oviducal tissue suspended in Ham's F10 + 10% fetal calf serum (F10FCS) or in F10FCS alone. After 4 days, the proportion of embryos developing into compact morulae or blastocysts was greater (P less than 0.005) in co-culture (38/82; 46%) than in F10FCS (1/27; 4%). In Exp. 2, a solution of collagenase, trypsin, DNAse and EDTA was used to disperse oviducal tissue, which was then cultured in TCM199 + 10% fetal calf serum (M199FCS) to obtain monolayers. Embryos (1-8 cells) were then co-cultured with monolayers or in M199FCS alone. The proportion of embryos developing into compact morulae and blastocysts after 4-5 days was higher (P less than 0.005) in co-culture (15/34; 43%) than in M199FCS (1/37; 3%); mean numbers of cells/embryo were also higher (P less than 0.001) (27.70; range 2-82 in co-culture; 8.83; range 2-18 in M199FCS). In Exp. 3, embryos obtained from in-vitro maturation and fertilization were used to compare development between co-culture and medium conditioned by oviducal tissue. Initial cleavage rate (no. embryos greater than 1 cell/total) was 76% (611/807) and did not differ among treatments. After 5 days, the proportion cleaving to greater than 16 cells was higher (P less than 0.005) in co-culture (71/203; 35%) and conditioned medium (48/205; 23%) compared to M199FCS (14/203; 7%). Similarly, the proportion developing into compact morulae and blastocysts was greater (P less than 0.005) in co-culture (44/203; 22%) and conditioned medium (46/205; 22%) than in M199FCS (7/203; 3%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2704004

  10. The modulation of tissue-specific gene expression in rat nasal chondrocyte cultures by bioactive glasses.

    PubMed

    Asselin, Audrey; Hattar, Susan; Oboeuf, Martine; Greenspan, David; Berdal, Ariane; Sautier, Jean-Michel

    2004-11-01

    Since bone repair may occur, following endochondral ossification, we have investigated the behaviour of chondrocytes isolated from nasal septum cartilage of foetal rats and cultured up to 21 days in the presence of a melt-derived bioactive glass (Bioglass 45S5) and a less reactive glass with 60 wt% silica content (60S). In both cultures, chondrocytes proliferate and form typical cartilaginous nodules on day 5 of cultures. However, on day 12, the nodules in contact with 45S5 granules became darker than in 60S cultures, corresponding to the emergence of matrix biomineralization. Transmission electron microscopy showed a collagen-rich matrix composed of densely packed fibres and mineralized foci formed of needle-shaped crystals in contact with an electron-dense layer located at the periphery of the material. The specific activity of alkaline phosphatase was significant higher in 45S5 cultures on day 15 than in 60S cultures. Real time RT-PCR was used to monitor gene expression levels of specific chondrogenic markers. The transcription factor Sox9 was expressed throughout the culture period, but with no significant differences between the two kinds of cultures. In contrast, Runx2 expression was higher in experiment cultures on day 12. Type II collagen mRNA and aggrecan, showed an almost similar expression pattern with a strong expression at the beginning of cultures but higher in experiment cultures. Indian hedgehog was strongly expressed between day 9 and 12 with a significant stimulation in 45S5 cultures. Similarly, type X collagen mRNA seemed to be up-regulated in 45S5 cultures on day 20. In conclusion, this study shows hat 45S5 Bioglass has the ability to support the growth of chondrocytes and to stimulate some chondrogenic molecular markers. PMID:15159078

  11. Insect transgenesis and the sterile insect technique

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The establishment of broadly applicable insect transgenesis systems will enable the analyses of gene function in diverse insect species. This will greatly increase our understanding of diverse aspects of biology so far not functionally addressable. Moreover, insect transgenesis will provide novel st...

  12. What Makes an Insect an Insect?

    ERIC Educational Resources Information Center

    NatureScope, 1985

    1985-01-01

    Provides background information on characteristics common to all insects, activities, and student materials (ready-to-copy games, puzzles, coloring pages, worksheets, and/or mazes) which describe: how insects are classified; how they are different from other animals; and the main insect characteristics. Activities include recommended age levels,…

  13. In vitro M-like cells genesis through a tissue-engineered triple-culture intestinal model.

    PubMed

    Araújo, Francisca; Pereira, Carla; Costa, Joana; Barrias, Cristina; Granja, Pedro L; Sarmento, Bruno

    2016-05-01

    Although fewer in number, M-cells are considered antigen sampling cells, acting as a gateway for antigens from the gut lumen and presenting an impressive aptitude for particle transcytosis. These features make M-cells attractive targets for oral drug delivery studies, but this has been poorly explored. New and reproducible tissue-like in vitro models for studying intestinal sampling and permeability mechanisms are needed. The combination of different cell players in such models offers improved microenvironments with higher physiologic relevance. Here, a tissue-engineered model was established, by co-culturing Caco-2 absorptive cells, HT29-MTX mucus-producing cells and Raji B lymphocytes. After 3 weeks of cell co-culture, the presence of M-like cells was evidenced by the loss of brush-border organization, detected by the lack of microvilli. The triple-culture model showed to be efficient for insulin transport, a process that was influenced by the tightness of junctions between epithelial cells and the presence of mucus and M-like cells. Ultimately, the proposed tissue-engineered model provides a more complete and reliable tool to perform drug permeability tests, as compared to traditional models, and may also find applicability as an in vitro system to study transdifferentiation mechanisms of M cells. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 104B:782-788, 2016. PMID:26313639

  14. Depletion of praziquantel in plasma and muscle tissue of cultured rockfish Sebastes schlegeli after oral and bath treatment.

    PubMed

    Kim, K H; Kim, C S; Kim, J W

    2001-08-01

    Depletion of praziquantel in plasma and muscle tissue after oral and bath treatments was studied in cultured rockfish Sebastes schlegeli. In the oral treatment, a single dose of 400 mg praziquantel kg(-1) body weight was administered by intubation of the stomach. A bath treatment at 100 ppm of praziquantel for 4 min was also carried out. Plasma and muscle tissue samples were collected at 3, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h post-treatment, and analyzed for praziquantel by reversed-phase HPLC using diazepam as the internal standard. Following oral treatment, praziquantel was detected in plasma and muscle tissue until 96 h after treatment. In plasma the praziquantel concentration was highest at the 9 h sampling time and declined sharply at the 48 h sampling point. The concentrations of praziquantel in the muscle tissue were lower than those in the plasma, and the highest value was found at the 9 h sampling time. Following bath treatment, praziquantel was found in plasma and muscle tissue until 72 and 24 h after treatment, respectively. In plasma the praziquantel concentration was highest at the 12 h sampling time and declined sharply thereafter. The concentrations of praziquantel in the muscle tissue were significantly lower than those in the plasma, and the concentrations declined consistently with time. PMID:11558729

  15. Estimation of dynamic metabolic activity in micro-tissue cultures from sensor recordings with an FEM model.

    PubMed

    Pfister, Cornelia; Forstmeier, Christian; Biedermann, Johannes; Schermuly, Julia; Demmel, Franz; Wolf, Peter; Kaspers, Bernd; Brischwein, Martin

    2016-05-01

    We estimated the dynamic cell metabolic activity and the distribution of the pH value and oxygen concentration in tissue samples cultured in vitro by using real-time sensor records and a numerical simulation of the underlying reaction-diffusion processes. As an experimental tissue model, we used chicken spleen slices. A finite element method model representing the biochemical processes and including the relevant sensor data was set up. By fitting the calculated results to the measured data, we derived the spatiotemporal values of the pH value, the oxygen concentration and the absolute metabolic activity (extracellular acidification and oxygen uptake rate) of the samples. Notably, the location of the samples in relation to the sensors has a great influence on the detectable metabolic rates. The long-term vitality of the tissue samples strongly depends on their size. We further discuss the benefits and limitations of the model. PMID:26296800

  16. NMR-based metabolomics study of the biochemical relationship between sugarcane callus tissues and their respective nutrient culture media

    PubMed Central

    Mahmud, Iqbal; Thapaliya, Monica; Boroujerdi, Arezue; Chowdhury, Kamal

    2014-01-01

    The culture of sugarcane leaf explant onto culture induction medium triggers the stimulation of cell metabolism into both embryogenic and non-embryogenic callus tissues. Previous analyses demonstrated that embryogenic and nonembryogenic callus tissues have distinct metabolic profiles. This study is the follow-up to understand the biochemical relationship between the nutrient media and callus tissues using one-dimensional (1D 1H) and two-dimensional (2D 1H–13C) NMR spectroscopy followed by principal component analysis (PCA). 1D 1H spectral comparisons of fresh unspent media (FM), embryogenic callus media (ECM), non-embryogenic callus media (NECM), embryogenic callus (EC), and non-embryogenic callus (NEC), showed different metabolic relationships between callus tissues and media. Based on metabolite fold change analysis, significantly changing sugar compounds such as glucose, fructose, sucrose, and maltose were maintained in large quantities by EC only. Significantly different amino acid compounds such as valine, leucine, alanine, threonine, asparagine, and glutamine and different organic acid derivatives such as lactate, 2-hydroxyisobutyrate, 4-aminobutyrate, malonate, and choline were present in EC, NEC, and NECM, which indicates that EC maintained these nutrients, while NEC either maintained or secreted the metabolites. These media and callus-specific results suggest that EC and NEC utilize and/or secrete media nutrients differently. PMID:25012359

  17. Influence of electrical stimulation on 3D-cultures of adipose tissue derived progenitor cells (ATDPCs) behavior.

    PubMed

    Castells-Sala, C; Sanchez, B; Recha-Sancho, L; Puig, V; Bragos, R; Semino, C E

    2012-01-01

    Tissue engineering has a fundamental role in regenerative medicine. Still today, the major motivation for cardiac regeneration is to design a platform that enables the complete tissue structure and physiological function regeneration of injured myocardium areas. Although tissue engineering approaches have been generally developed for two-dimensional (2D) culture systems, three-dimensional (3D) systems are being spotlighted as the means to mimic better in vivo cellular conditions. This manuscript examines the influence of electrical stimulation on 3D cultures of adipose tissue-derived progenitor cells (ATDPCs). ATDPCs cells were encapsulated into a self-assembling peptide nanoscaffold (RAD16-I) and continuously electro stimulated during 14-20 days with 2-ms pulses of 50mV/cm at a frequency of 1 Hz. Good cellular network formation and construct diameter reduction was observed in electro stimulated samples. Importantly, the process of electro stimulation does not disrupt cell viability or connectivity. As a future outlook, differentiation studies to cardiomyocytes-like cells will be performed analyzing gene profile and protein expression. PMID:23367213

  18. Irreversible damage in ovine ovarian tissue after cryopreservation in propanediol: analyses after in vitro culture and xenotransplantation.

    PubMed

    Oskam, I C; Lund, T; Santos, R R

    2011-10-01

    Current progress in cancer treatment has increased the incidence of long-term patient survival. Ovarian tissue cryopreservation (OT) is still the most promising fertility saving method offered to young female patients with cancer prior to the onset of radio-chemotherapy. Further follicular development of immature primordial follicles depends on transplantation or in vitro culture (IVC). Aim of this study was to evaluate the appropriateness of cryopreserved ovine OT with 1,2-propanediol (PROH) after short-term IVC and xenotransplantation (XT). Ovarian tissue fragments from young adult sheep were cryopreserved using a standard slow-freezing protocol with 1.5 M PROH. Cryopreserved OT was assessed by light- and transmission electron microscopic analyses after thawing, IVC or XT in severe immunodeficient mice. Control OT showed the presence of healthy preantral follicles (Mean: 78.8%; SE 2.9%) and normal structure of the stromal tissue. After thawing and IVC over 80% of damaged primordial follicles and poor preservation of the stromal tissue was observed. After XT, OT demonstrated deficient follicles and huge areas of vacuolization in the stromal tissue confirmed by ultrastructural assessment. In conclusion, because of the irreversible character of the follicular and stromal damage of cryopreserved ovine ovarian tissue after IVC and XT, strong improvement of the utilized protocol is needed to be suitable for the preservation of ovine ovarian tissue. The deleterious effects of PROH do not imply its exclusion as cryoprotectant, but more research is needed for the development of less toxic cryoprotectant mixtures and toxicity neutralizers with attested cryoprotectant capacity for the safe and feasible freezing of human ovarian tissue. PMID:21272097

  19. Tissue-specific Cd and Pb accumulation in Peruvian scallop (Argopecten purpuratus) transplanted to a suspended and bottom culture at Sechura Bay, Peru.

    PubMed

    Loaiza, Iván; Hurtado, Daniela; Miglio, Maria; Orrego, Henry; Mendo, Jaime

    2015-02-28

    In order to understand the effect of different culture systems on Cd and Pb accumulation, suspended long-line and bottom cultures of Argopecten purpuratus were conducted during January until April 2010 (120 days). The Cd tissue levels were the highest at the middle of the experiment (30-d till 70-d) for suspended-cultured individuals, while bottom-cultured individuals showed an increasing trend. Gonad Pb levels were also higher during the same period for all cultures, while adductor muscle exhibited no considerable variations. Cd and Pb tissue concentrations were mainly greater in deeper cultures. There were no significant differences in Cd and Pb accumulation between individual sizes. The Cd and Pb levels in edible tissue (gonad+adductor muscle) did not exceed the EU and FDA maximum levels. Based on the target hazard quotient (THQ) and the provisional tolerance weekly intake (PTWI), no risk (THQ<1 and %PTWI<30) was found for human consumption. PMID:25444617

  20. Line following terrestrial insect biobots.

    PubMed

    Latif, Tahmid; Bozkurt, Alper

    2012-01-01

    The present day technology falls short in offering centimeter scale mobile robots that can function effectively under unknown and dynamic environmental conditions. Insects, on the other hand, exhibit an unmatched ability to navigate through a wide variety of environments and overcome perturbations by successfully maintaining control and stability. In this study, we use neural stimulation systems to wirelessly navigate cockroaches to follow lines to enable terrestrial insect biobots. We also propose a system-on-chip based ZigBee enabled wireless neurostimulation backpack system with on-board tissue-electrode bioelectrical coupling verification. Such a capability ensures an electrochemically safe stimulation and avoids irreversible damage to the interface which is often misinterpreted as habituation of the insect to the applied stimulation. PMID:23366056

  1. Book Review: Insect Virology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Viruses that infect insects have long been of interest both as a means for controlling insect pest populations in an environmentally safe manner, and also as significant threats to beneficial insects of great value, such as honey bees and silkworms. Insect viruses also have been of intrinsic intere...

  2. Insect-ual Pursuits.

    ERIC Educational Resources Information Center

    Mallow, David

    1991-01-01

    Explains how insects can be used to stimulate student writing. Describes how students can create their own systems to classify and differentiate insects. Discusses insect morphology and includes three detailed diagrams. The author provides an extension activity where students hypothesize about the niche of an insect based on its anatomy. (PR)

  3. Rate of change in central corneal thickness: a viability indicator for conventional drainage tissues in organ culture.

    PubMed

    Wan, Z; Brigatti, L; Ranger-Moore, J; Ethier, C R; Stamer, W D

    2006-06-01

    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. PMID:16466713

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

    PubMed Central

    Kiyomi, Anna; Makita, Masujiro; Ozeki, Tomoko; Li, Na; Satomura, Aiko; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Iwase, Takuji; Hirano, Toshihiko

    2015-01-01

    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 < IIB (P = 0.0096). CONCLUSIONS: The tumor 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. PMID:26310378

  5. Influence of Culture Conditions and Extracellular Matrix Alignment on Human Mesenchymal Stem Cell Invasion into Decellularized Engineered Tissues

    PubMed Central

    Weidenhamer, Nathan K.; Moore, Dusty L.; Lobo, Fluvio L.; Klair, Nathaniel T.; Tranquillo, Robert T.

    2015-01-01

    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 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 remodeling using circular and C-shaped molds. 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, hMSC 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 be differentiating toward a smooth muscle cell or myofibroblast phenotype with ascorbic acid treatment. These results inform the strategy of recellularizing decellularized engineered tissue with hMSCs. PMID:25556358

  6. Reconstruction of conjunctival epithelium-like tissue using a temperature-responsive culture dish

    PubMed Central

    Yao, Qinke; Zhu, Mengyu; Chen, Junzhao; Shao, Chunyi; Yan, Chenxi; Wang, Zi; Fan, Xianqun; Gu, Ping

    2015-01-01

    Purpose To study the feasibility of engineering conjunctival epithelial cell sheets on a temperature-responsive culture dish for ocular surface reconstruction. Methods Rabbit conjunctival epithelial cells (rCjECs) were cultured in DMEM/F-12 (1:1) medium. The morphology and phenotype of the rCjECs were confirmed with phalloidin staining, periodic acid–Schiff (PAS) staining, and immunocytochemistry. The rCjECs cultured on a temperature-responsive culture dish for 10 days produced confluent conjunctival epithelial cell sheets. Then, the phenotype, structure, and function of the conjunctival epithelial cell sheets were examined. Results The conjunctival epithelial cells were compact, uniform, and cobblestone shape. All cultured conjunctival epithelial cells were harvested as intact cell sheets by reducing the culture temperature to 20 °C. Conjunctival epithelial cells were stratified in four to five cell layers similar to the conjunctival epithelium. CCK-8 analysis, 5-bromo-2’-deoxyuridine (BrdU) staining, and the live and dead viability assay confirmed that viable proliferation cells were retained in the cell sheets. Immunohistochemistry for CK4, CK19, and MUC5AC showed the cell sheets still maintained characteristics of the conjunctival epithelium. Conclusions A temperature-responsive culture dish enables fabrication of viable conjunctival epithelial cell sheets with goblet cells and proliferative cells. Conjunctival epithelial cell sheets will be promising for reconstruction of the conjunctival epithelium. PMID:26396489

  7. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  8. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    PubMed Central

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Wu, Jichuan; Hubert, Terrence L.; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  9. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    PubMed

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  10. Enhanced Adhesion and OspC Protein Synthesis of the Lyme Disease Spirochete Borrelia Burgdorferi Cultivated in a Host-Derived Tissue Co-Culture System

    PubMed Central

    Şen, Ece; Sigal, Leonard H.

    2013-01-01

    Background: The adhesion process of Borrelia burgdorferi to susceptible host cell has not yet been completely understood regarding the function of OspA, OspB and OspC proteins and a conflict exists in the infection process. Aims: The adhesion rates of pathogenic (low BSK medium passaged or susceptible rat joint tissue co-cultivated) or non-pathogenic Borrelia burgdorferi (high BSK medium passaged) isolate (FNJ) to human umbilical vein endothelial cells (HUVEC) cultured on coverslips and the synthesis of OspA and OspC proteins were investigated to analyze the infection process of this bacterium. Study Design: In-vitro study. Methods: Spirochetes were cultured in BSK medium or in a LEW/N rat tibiotarsal joint tissue feeder layer supported co-culture system using ESG co-culture medium and labelled with 3H-adenine for 48 hours. SDS-PAGE, Western Blotting, Immunogold A labeling as well as radiolabeling experiments were used to compare pathogenic or non pathogenic spirochetes during the adhesion process. Results: Tissue co-cultured B. burgdorferi adhered about ten times faster than BSK-grown spirochetes. Trypsin inhibited attachment to HUVEC and co-culture of trypsinized spirochetes with tissues reversed the inhibition. Also, the synthesis of OspC protein by spirochetes was increased in abundance after tissue co-cultures, as determined by SDS-PAGE and by electron microscopy analysis of protein A-immunogold staining by anti-OspC antibodies. OspA protein was synthesized in similar quantities in all Borrelia cultures analyzed by the same techniques. Conclusion: Low BSK passaged or tissue co-cultured pathogenic Lyme disease spirochetes adhere to HUVEC faster than non-pathogenic high BSK passaged forms of this bacterium. Spirochetes synthesized OspC protein during host tissue-associated growth. However, we did not observe a reduction of OspA synthesis during host tissue co-cultivation in vitro. PMID:25207103

  11. Technical and theoretical considerations about gradient perfusion culture for epithelia used in tissue engineering, biomaterial testing and pharmaceutical research.

    PubMed

    Minuth, Will W; Strehl, Raimund

    2007-06-01

    Epithelia act as biological barriers, which are exposed to different environments at the luminal and basal sides. To simulate this situation and to improve functional features an in vitro gradient perfusion culture technique was developed in our laboratory. This innovative technique appears to be simple at first sight, but the performance needs practical and theoretical knowledge. To harvest intact epithelia after a long-term gradient culture period of many days, leakage, edge damage and pressure differences in the system have to be avoided so that the epithelial barrier function is maintained continuously. Unexpectedly, one of the major obstacles are micro-injuries in the epithelia caused by gas bubbles, which arise during transportation of the medium or due to respiration of the cultured tissue. Gas bubbles randomly accumulate either at the luminal or basal fluid flow of the gradient perfusion culture container. This phenomenon results in fluid pressure differences between the luminal and basal perfusion compartments of the gradient container, which in turn leads to damage of the barrier function. Consequently, the content of gas bubbles in the transported culture medium has to be minimized. Thus, our technical concept is the reduction of gas bubbles while keeping the content of oxygen constant. To follow this strategy we developed a new type of screw cap for media bottles specifically designed to allow fluid contact only with tube and not with cap material. Furthermore, a gas expander module separates gas bubbles from the liquid phase during transportation of the medium. Finally, a new type of gradient culture container allows a permanent elimination of transported gas bubbles. Application of this innovative equipment optimizes the parallel transportation of fluid in the luminal and basal compartments of a gradient culture container. PMID:18458434

  12. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    EPA Science Inventory

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  13. Influence of scaffold morphology on co-cultures of human endothelial and adipose tissue-derived stem cells.

    PubMed

    Arnal-Pastor, M; Martínez-Ramos, C; Vallés-Lluch, A; Pradas, M Monleón

    2016-06-01

    The interior of tissue engineering scaffolds must be vascularizable and allow adequate nutrients perfusion in order to ensure the viability of the cells colonizing them. The promotion of rapid vascularization of scaffolds is critical for thick artificial constructs. In the present study co-cultures of human endothelial and adipose tissue-derived stem cells have been performed in poly(ethyl acrylate) scaffolds with two different pore structures: grid-like (PEA-o) or sponge-like (PEA-s), in combination with a self-assembling peptide gel filling the pores, which aims to mimic the physiological niche. After 2 and 7 culture days, cell adhesion, proliferation and migration, the expression of cell surface markers like CD31 and CD90 and the release of VEGF were assessed by means of immunocytochemistry, scanning electronic microscopy, flow cytometry and ELISA analyses. The study demonstrated that PEA-s scaffolds promoted greater cell organization into tubular-like structures than PEA-o scaffolds, and this was enhanced by the presence of the peptide gel. Paracrine signaling from adipose cells significantly improved endothelial cell viability, proving the advantageous combination of this system for obtaining easily vascularizable tissue engineered grafts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1523-1533, 2016. PMID:26860551

  14. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue

    SciTech Connect

    McCarthy, K.D.; de Vellis, J.

    1980-06-01

    A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15 to 18 h at 37/sup 0/C. Preparations appear >98% pure and contain approx.1-2 x 10/sup 7/ viable cells (20 to 40 mg of cell protein). Three methods were used to characterize these two culture types. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markets, 2',3' -cycle nucleotide 3' -phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrasturcture of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.

  15. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    PubMed

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies. PMID:25636587

  16. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  17. Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues

    PubMed Central

    HU, PENGFEI; PU, YABIN; LI, XIAYUN; ZHU, ZHIQIANG; ZHAO, YUHUA; GUAN, WEIJUN; MA, YUEHUI

    2015-01-01

    Lung-derived mesenchymal stem cells (LMSCs) are considered to be important in lung tissue repair and regenerative processes. However, the biological characteristics and differentiation potential of LMSCs remain to be elucidated. In the present study, fetal lung-derived mesenchymal stem cells (FLMSCs) were isolated from fetal bovine lung tissues by collagenase digestion. The in vitro culture conditions were optimized and stabilized and the self-renewal ability and differentiation potential were evaluated. The results demonstrated that the FLMSCs were morphologically consistent with fibroblasts, were able to be cultured and passaged for at least 33 passages and the cell morphology and proliferative ability were stable during the first 10 passages. In addition, FLMSCs were found to express CD29, CD44, CD73 and CD166, however, they did not express hematopoietic cell specific markers, including CD34, CD45 and BOLA-DRα. The growth kinetics of FLMSCs consisted of a lag phase, a logarithmic phase and a plateau phase, and as the passages increased, the proliferative ability of cells gradually decreased. The majority of FLMSCs were in G0/G1 phase. Following osteogenic induction, FLMSCs were positive for the expression of osteopontin and collagen type I α2. Following neurogenic differentiation, the cells were morphologically consistent with neuronal cells and positive for microtubule-associated protein 2 and nestin expression. It was concluded that the isolated FLMSCs exhibited typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their proliferation and the maintenance of stemness. The present study illustrated the potential application of lung tissue as an adult stem cell source for regenerative therapies. PMID:26016556

  18. Growth of plant tissue cultures in simulated lunar soil: Implications for a lunar base CELSS (Controlled Ecological Life Support System)

    NASA Technical Reports Server (NTRS)

    Venketeswaran, S.

    1988-01-01

    Experiments were carried out on plant tissue cultures, seed germination, seedling development and plants grown on Simulated Lunar Soil to evaluate the potential of future development of lunar based agriculture. The studies done to determine the effect of the placement of SLS on tissue cultures showed no adverse effect of SLS on tissue cultures. Although statistically insignificant, SLS in suspension showed a comparatively higher growth rate. Observations indicate the SLS, itself cannot support calli growth but was able to show a positive effect on growth rate of calli when supplemented with MS salts. This positive effect related to nutritive value of the SLS was found to have improved at high pH levels, than at the recommended low pH levels for standard media. Results from seed germination indicated that there is neither inhibitory, toxicity nor stimulatory effect of SLS, even though SLS contains high amounts of aluminum compounds compared to earth soil. Analysis of seeding development and growth data showed significant reduction in growth rate indicating that, SLS was a poor growth medium for plant life. This was confirmed by the studies done with embryos and direct plant growth on SLS. Further observations attributed this poor quality of SLS is due to it's lack of essential mineral elements needed for plant growth. By changing the pH of the soil, to more basic conditions, the quality of SLS for plant growth could be improved up to a significant level. Also it was found that the quality of SLS could be improved by almost twice, by external supply of major mineral elements, directly to SLS.

  19. A Silk Fibroin and Peptide Amphiphile-Based Co-Culture Model for Osteochondral Tissue Engineering.

    PubMed

    Çakmak, Soner; Çakmak, Anıl S; Kaplan, David L; Gümüşderelioğlu, Menemşe

    2016-08-01

    New biomaterials with the properties of both bone and cartilage extracellular matrices (ECM) should be designed and used with co-culture systems to address clinically applicable osteochondral constructs. Herein, a co-culture model is described based on a trilayered silk fibroin-peptide amphiphile (PA) scaffold cultured with human articular chondrocytes (hACs) and human bone marrow mesenchymal stem cells (hBMSCs) in an osteochondral cocktail medium for the cartilage and bone sides, respectively. The presence of hACs in the co-cultures significantly increases the osteogenic differentiation potential of hBMSCs based on ALP activity, RT-PCR for osteogenic markers, calcium analyses, and histological stainings, whereas hACs produces a significant amount of glycosaminoglycans (GAGs) for the cartilage region, even in the absence of growth factor TGF-β family in the co-culture medium. This trilayered scaffold with trophic effects offers a promising strategy for the study of osteochondral defects. PMID:27139244

  20. Studies on the medicinal properties of Solanum chrysotrichum in tissue culture: I. Callus formation and plant induction from axillary buds.

    PubMed

    Villarreal, M L; Muñoz, J

    1991-01-01

    A tissue culture method is described for micropropagation and callus formation from Solanum chrysotricum axillary bud explants in Murashige and Skoog's (MS) medium, supplemented with various growth regulators. Induction of rooted plants were initiated only when indol-3 acetic acid (IAA) was present as an auxin in combination with either of two cytokinins: kinetin (KN) or benzyladenine (BA); however, the combination of IAA (0.1 mg.lt.-1) + BA (0.2 mg.lt.-1) was found to be best suited for morphogenesis purposes. Alternatively, callus tissue formation was influenced in presence of naphthalene acetic acid; which in combination with kinetin (NAA 0.1 mg.lt.-1 + KN 0.2 mg.lt.-1) exhibit the best response studied. The plant material obtained by this procedure is proposed for pharmacological and chemical studies of this important antimycotic plant remedy. PMID:1819987

  1. Composite Scaffolds Containing Silk Fibroin, Gelatin, and Hydroxyapatite for Bone Tissue Regeneration and 3D Cell Culturing

    PubMed Central

    Moisenovich, M. M.; Arkhipova, A. Yu.; Orlova, A. A.; Drutskaya, M. S; Volkova, S. V.; Zacharov, S. E.; Agapov, I. I.; Kirpichnikov, M. P.

    2014-01-01

    Three-dimensional (3D) silk fibroin scaffolds were modified with one of the major bone tissue derivatives (nano-hydroxyapatite) and/or a collagen derivative (gelatin). Adhesion and proliferation of mouse embryonic fibroblasts (MEF) within the scaffold were increased after modification with either nano-hydroxyapatite or gelatin. However, a significant increase in MEF adhesion and proliferation was observed when both additives were introduced into the scaffold. Such modified composite scaffolds provide a new and better platform to study wound healing, bone and other tissue regeneration, as well as artificial organ bioengineering. This system can further be applied to establish experimental models to study cell-substrate interactions, cell migration and other complex processes, which may be difficult to address using the conventional two-dimensional culture systems. PMID:24772332

  2. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

    PubMed

    Cavalcante, A Y P; Gouveia, B B; Barberino, R S; Lins, T L B G; Santos, L P; Gonçalves, R J S; Celestino, J J H; Matos, M H T

    2016-08-01

    This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue. PMID:26503557

  3. Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones

    PubMed Central

    De Block, Marc

    1990-01-01

    Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25°C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO3−/NH4+ ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba × P. tremula and of the poplar hybrid Populus trichocarpa × P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not. Images Figure 1 Figure 2 Figure 4 PMID:16667565

  4. Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

    PubMed

    Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

    2014-11-01

    The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity. PMID:25142347

  5. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    SciTech Connect

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

  6. Tissue dynamics spectroscopy for phenotypic profiling of drug effects in three-dimensional culture.

    PubMed

    Nolte, David D; An, Ran; Turek, John; Jeong, Kwan

    2012-11-01

    Coherence-gated dynamic light scattering captures cellular dynamics through ultra-low-frequency (0.005-5 Hz) speckle fluctuations and Doppler shifts that encode a broad range of cellular and subcellular motions. The dynamic physiological response of tissues to applied drugs is the basis for a new type of phenotypic profiling for drug screening on multicellular tumor spheroids. Volumetrically resolved tissue-response fluctuation spectrograms act as fingerprints that are segmented through feature masks into high-dimensional feature vectors. Drug-response clustering is achieved through multidimensional scaling with simulated annealing to construct phenotypic drug profiles that cluster drugs with similar responses. Hypoxic vs. normoxic tissue responses present two distinct phenotypes with differentiated responses to environmental perturbations and to pharmacological doses. PMID:23162721

  7. Understanding cross-communication between aboveground and belowground tissues via transcriptome analysis of a sucking insect whitefly-infested pepper plants.

    PubMed

    Park, Yong-Soon; Ryu, Choong-Min

    2014-01-01

    Plants have developed defensive machinery to protect themselves against herbivore and pathogen attacks. We previously reported that aboveground whitefly (Bemisia tabaci Genn.) infestation elicited induced resistance in leaves and roots and influenced the modification of the rhizosphere microflora. In this study, to obtain molecular evidence supporting these plant fitness strategies against whitefly infestation, we performed a 300 K pepper microarray analysis using leaf and root tissues of pepper (Capsicum annuum L.) applied with whitefly, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), and the combination of BTH+whitefly. We defined differentially expressed genes (DEGs) as genes exhibiting more than 2-fold change (1.0 based on log2 values) in expression in leaves and roots in response to each treatment compared to the control. We identified a total of 16,188 DEGs in leaves and roots. Of these, 6685, 6752, and 4045 DEGs from leaf tissue and 6768, 7705, and 7667 DEGs from root tissue were identified in the BTH, BTH+whitefly, and whitefly treatment groups, respectively. The total number of DEGs was approximately two-times higher in roots than in whitefly-infested leaves subjected to whitefly infestation. Among DEGs, whitefly feeding induced salicylic acid and jasmonic acid/ethylene-dependent signaling pathways in leaves and roots. Several transporters and auxin-responsive genes were upregulated in roots, which can explain why biomass increase is facilitated. Using transcriptome analysis, our study provides new insights into the molecular basis of whitefly-mediated intercommunication between aboveground and belowground plant tissues and provides molecular evidence that may explain the alteration of rhizosphere microflora and root biomass by whitefly infestation. PMID:24309116

  8. Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

    NASA Astrophysics Data System (ADS)

    Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.

    2015-03-01

    Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.

  9. Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

    PubMed

    Sales, A D; Duarte, A B G; Santos, R R; Alves, K A; Lima, L F; Rodrigues, G Q; Brito, I R; Lobo, C H; Bruno, J B; Locatelli, Y; Figueiredo, J R; Rodrigues, A P R

    2016-08-01

    Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality. PMID:26975215

  10. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  11. Plant Tissue Culture Development and Biotechnology, Chapter 10: Molecular Tools for Studying Plant Genetic Diversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitous nature of DNA is a central theme for all biology. The nucleus of each cell that makes up an organism contains genomic DNA, which is the blueprint for life. The differential expression of genes within each cell gives rise to different tissues, organs and, ultimately, different organism...

  12. 3D BREAST TISSUE CO-CULTURES FOR SCREENING MAMMARY CARCINOGENS - PHASE I

    EPA Science Inventory

    Breast cancer is not a disease of individual cells, but principally a failure of cells and tissues to communicate properly. One communication mechanism that is frequently disrupted in breast cancer involves the hormone estrogen. Despite recognition that exposure to compound...

  13. State Vector Description of the Proliferation of Mammalian Cells in Tissue Culture

    PubMed Central

    Hahn, George M.

    1966-01-01

    Proliferation of mammalian cells, even under conditions of unlimited growth, presents a complex problem because of the interaction of deterministic and stochastic processes. Division of the cell cycle into a finite number of parts establishes a multidimensional vector space. In this space an arbitrary culture can be represented by a vector called the state vector. The culture's subsequent growth is represented mathematically as a series of transformations of the state vector. The operators effecting these transformations are presented in matrix form and their relationship to the distribution of cell generation times is described. As an application of the model, the growth of an initially synchronized culture is considered and an unambiguous measure of the degree of synchrony is proposed. Results of a computer simulation of such a culture show the behavior with time of the degree of synchrony, the total cell number, and the mitotic index. In particular the importance of the magnitude of the coefficient of variation of the generation time distribution is illustrated. PMID:5963460

  14. COMPARISON OF TISSUE CULTURE AND ANIMAL MODELS FOR ASSESSMENT OF CRYPTOSPRIDIUM PARVUM INFECTION

    EPA Science Inventory

    Data from three different disinfection studies using both cell culture and mouse infectivity to assess Cryptosporidium parvum inactivation were evaluated in a total of 35 comparison including process controls and treated samples. C. parvum infectivity in the in vitro FDM-MPN assa...

  15. Use of HyperCard to Simulate a Tissue Culture Laboratory.

    ERIC Educational Resources Information Center

    Nester, Bradley S.; Turney, Tully H.

    1992-01-01

    Describes the use of a Macintosh computer and HyperCard software to create an introduction to cell culture techniques that closely approximates a hands-on laboratory experiment. Highlights include data acquisition, data analysis, the generation of growth curves, and electronic modeling. (LRW)

  16. Amending storage vessel and media improves transfer interval of Musa spp. tissue culture plantlets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Musa spp. are some of the most important fruit food crops in the world. The USDA-ARS TARS maintains a Musa spp. germplasm collection of ~150 accessions in field plots and in medium-term storage in vitro. Accessions maintained in vitro require routine sub-culturing as nutrient medium is lost due to ...

  17. Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transfe...

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

    PubMed

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

    2015-04-01

    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. PMID:25626739

  19. TISSUE CULTURE AS A METHOD FOR EVALUATING THE BIOTRANSFORMATION OF XENOBIOTICS BY PLANTS

    EPA Science Inventory

    Suspension cultures of Rosa cv. Paul's scarlet were used as a model system to examine the metabolism of l,3-dinitrobenzene (DNB), an industrial waste compound. n a three-day period, 90% of the DNB supplied (96 nmoles) was metabolized by approximately 12 grams (fresh weight) of ce...

  20. Cortical radial glia: identification in tissue culture and evidence for their transformation to astrocytes.

    PubMed

    Culican, S M; Baumrind, N L; Yamamoto, M; Pearlman, A L

    1990-02-01

    Radial glia are transiently present in the developing cerebral cortex, where they are thought to guide the migration of neurons from the proliferative zone to the forming cortical plate. To provide a framework for experimental studies of radial glia, we have defined morphological and immunocytochemical criteria to identify them in primary cultures of cortical cells obtained at embryonic day 13 in the mouse. Cortical radial glia in culture for 1-2 d resemble radial glia in vivo: they have a long, thin, unbranched process extending from one or both ends of the elongated cell body and are labeled with the monoclonal antibody RC1 but not with antibodies to glial fibrillary acidic protein (abGFAP). We tested the specificity of RC1 by double-labeling with a panel of cell-type specific antibodies, and found that it labels radial glia, astrocytes, and fibroblast-like cells, but not neurons. Fibroblasts are easily distinguished from glia by morphology and by labeling with antibodies to fibronectin. To test the hypothesis that radial glia become astrocytes when their developmental role is complete, we examined their morphological and immunocytochemical development in culture. After 3-4 d in vitro radial glia develop several branched processes; in this transitional stage they are labeled by both RC1 and abGFAP. Many radial glia lose RC1 immunoreactivity as they become increasingly branched and immunoreactive to abGFAP. In areas of the cultures that have few neurons and in cultures depleted of neurons by washing, flat, nonprocess-bearing glia predominate. These cells do not lose immunoreactivity to RC1 during the 9-d period of observation even though they acquire GFAP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2303868

  1. Towards the elements of successful insect RNAi

    PubMed Central

    Scott, Jeffrey G.; Michel, Kristin; Bartholomay, Lyric; Siegfried, Blair D.; Hunter, Wayne B.; Smagghe, Guy; Zhu, Kun Yan; Douglas, Angela E.

    2013-01-01

    RNA interference (RNAi), the sequence-specific suppression of gene expression, offers great opportunities for insect science, especially to analyze gene function, manage pest populations, and reduce disease pathogens. The accumulating body of literature on insect RNAi has revealed that the efficiency of RNAi varies between different species, the mode of RNAi delivery, and the genes being targeted. There is also variation in the duration of transcript suppression. At present, we have a limited capacity to predict the ideal experimental strategy for RNAi of a particular gene/insect because of our incomplete understanding of whether and how the RNAi signal is amplified and spread among insect cells. Consequently, development of the optimal RNAi protocols is a highly empirical process. This limitation can be relieved by systematic analysis of the molecular physiological basis of RNAi mechanisms in insects. An enhanced conceptual understanding of RNAi function in insects will facilitate the application of RNAi for dissection of gene function, and to fast-track the application of RNAi to both control pests and develop effective methods to protect beneficial insects and non-insect arthropods, particularly the honey bee (Apis mellifera) and cultured Pacific white shrimp (Litopenaeus vannamei) from viral and parasitic diseases. PMID:24041495

  2. Insulin and insulin-like growth factor-1 induce pronounced hypertrophy of skeletal myofibers in tissue culture

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.; Karlisch, Patricia; Shansky, Janet

    1990-01-01

    Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a serum-free medium for at least 6 to 7 days when embedded in a three dimensional collagen gel matrix. The myofibers are metabolically sensitive to physiological concentrations of insulin but these concentrations do not stimulate cell growth. Higher insulin concentrations stimulate both cell hyperplasia and myofiber hypertrophy. Cell growth results from a long term 42 percent increase in total protein synthesis and a 38 percent increase in protein degradation. Myofiber diameters increase by 71 to 98 percent after 6 to 7 days in insulin-containing medium. Insulin-like growth factor-1 but not insulin-like growth factor-2, at 250 ng/ml, is as effective as insulin in stimulating cell hyperplasia and myofiber hypertrophy. This model system provides a new method for studying the long-term anabolic effects of insulin and insulin-like growth factors on myofiber hypertrophy under defined tissue culture conditions.

  3. Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

    PubMed Central

    Keslová-Veselíková, Jana; Hůlková, Helena; Dobrovolný, Robert; Asfaw, Befekadu; Poupětová, Helena; Berná, Linda; Sikora, Jakub; Goláň, Lubor

    2008-01-01

    The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage. PMID:18351385

  4. Osteogenic potential of human adipose-tissue-derived mesenchymal stromal cells cultured on 3D-printed porous structured titanium.

    PubMed

    Lewallen, Eric A; Jones, Dakota L; Dudakovic, Amel; Thaler, Roman; Paradise, Christopher R; Kremers, Hilal M; Abdel, Matthew P; Kakar, Sanjeev; Dietz, Allan B; Cohen, Robert C; Lewallen, David G; van Wijnen, Andre J

    2016-05-01

    Integration of porous metal prosthetics, which restore form and function of irreversibly damaged joints, into remaining healthy bone is critical for implant success. We investigated the biological properties of adipose-tissue-derived mesenchymal stromal/stem cells (AMSCs) and addressed their potential to alter the in vitro microenvironment of implants. We employed human AMSCs as a practical source for musculoskeletal applications because these cells can be obtained in large quantities, are multipotent, and have trophic paracrine functions. AMSCs were cultured on surgical-grade porous titanium disks as a model for orthopedic implants. We monitored cell/substrate attachment, cell proliferation, multipotency, and differentiation phenotypes of AMSCs upon osteogenic induction. High-resolution scanning electron microscopy and histology revealed that AMSCs adhere to the porous metallic surface. Compared to standard tissue culture plastic, AMSCs grown in the porous titanium microenvironment showed differences in temporal expression for genes involved in cell cycle progression (CCNB2, HIST2H4), extracellular matrix production (COL1A1, COL3A1), mesenchymal lineage identity (ACTA2, CD248, CD44), osteoblastic transcription factors (DLX3, DLX5, ID3), and epigenetic regulators (EZH1, EZH2). We conclude that metal orthopedic implants can be effectively seeded with clinical-grade stem/stromal cells to create a pre-conditioned implant. PMID:26774799

  5. Three-Dimensional Culture of Cells and Matrix Biomolecules for Engineered Tissue Development and Biokinetics Model Validation

    PubMed Central

    Mason, Shelley S.; Kohles, Sean S.; Zelick, Randy D.; Winn, Shelley R.; Saha, Asit K.

    2011-01-01

    There has been considerable progress in cellular and molecular engineering due to recent advances in multiscale technology. Such technologies allow controlled manipulation of physiochemical interactions among cells in tissue culture. In particular, a novel chemomechanical bioreactor has recently been designed for the study of bone and cartilage tissue development, with particular focus on extracellular matrix formation. The bioreactor is equally significant as a tool for validation of mathematical models that explore biokinetic regulatory thresholds (Saha, A. K., and Kohles, S. S., 2010, “A Distinct Catabolic to Anabolic Threshold Due to Single-Cell Nanomechanical Stimulation in a Cartilage Biokinetics Model,” J. Nanotechnol. Eng. Med., 1(3), p. 031005; 2010, “Periodic Nanomechanical Stimulation in a Biokinetics Model Identifying Anabolic and Catabolic Pathways Associated With Cartilage Matrix Homeostasis,” J. Nanotechnol. Eng. Med., 1(4), p. 041001). In the current study, three-dimensional culture protocols are described for maintaining the cellular and biomolecular constituents within defined parameters. Preliminary validation of the bioreactor’s form and function, expected bioassays of the resulting matrix components, and application to biokinetic models are described. This approach provides a framework for future detailed explorations combining multiscale experimental and mathematical analyses, at nanoscale sensitivity, to describe cell and biomolecule dynamics in different environmental regimes. PMID:21709743

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

    PubMed Central

    Nodale, Cristina; D'Amici, Sirio; Maffucci, Diana; Ceccarelli, Simona; Monti, Marco; Benedetti Panici, Pierluigi; Romano, Ferdinando; Angeloni, Antonio; Marchese, Cinzia

    2014-01-01

    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. PMID:25162002

  7. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms.

    PubMed

    Loessner, Daniela; Meinert, Christoph; Kaemmerer, Elke; Martine, Laure C; Yue, Kan; Levett, Peter A; Klein, Travis J; Melchels, Ferry P W; Khademhosseini, Ali; Hutmacher, Dietmar W

    2016-04-01

    Progress in advancing a system-level understanding of the complexity of human tissue development and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of these processes in a physiological context. Hence, growing demand by cell biologists for organ-specific extracellular mimics has led to the development of a plethora of 3D cell culture assays based on natural and synthetic matrices. We developed a physiological microenvironment of semisynthetic origin, called gelatin methacryloyl (GelMA)-based hydrogels, which combine the biocompatibility of natural matrices with the reproducibility, stability and modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation of the GelMA polymer, which takes 1-2 weeks to complete, and which can be used to prepare hydrogel-based 3D cell culture models for cancer and stem cell research, as well as for tissue engineering applications. We also describe quality control and validation procedures, including how to assess the degree of GelMA functionalization and mechanical properties, to ensure reproducibility in experimental and animal studies. PMID:26985572

  8. Insect Bites and Stings

    MedlinePlus

    Most insect bites are harmless, though they sometimes cause discomfort. Bee, wasp, and hornet stings and fire ant bites usually hurt. Mosquito and flea bites usually itch. Insects can also spread diseases. In the United States, ...

  9. Insects: An Interdisciplinary Unit

    ERIC Educational Resources Information Center

    Leger, Heather

    2007-01-01

    The author talks about an interdisciplinary unit on insects, and presents activities that can help students practice communication skills (interpersonal, interpretive, and presentational) and learn about insects with hands-on activities.

  10. Respiration in Aquatic Insects.

    ERIC Educational Resources Information Center

    MacFarland, John

    1985-01-01

    This article: (1) explains the respiratory patterns of several freshwater insects; (2) describes the differences and mechanisms of spiracular cutaneous, and gill respiration; and (3) discusses behavioral aspects of selected aquatic insects. (ML)

  11. Insects and Scorpions

    MedlinePlus

    ... gov . Workplace Safety and Health Topics Insects & Scorpions Bees, Wasps, and Hornets Fire Ants Scorpions Additional Resources ... to outdoor workers. Stinging or biting insects include bees, wasps, hornets, and fire ants. The health effects ...

  12. Ecophysiology and insect herbivory

    SciTech Connect

    Clancy, K.M.; Wagner, M.R.; Reich, P.B.

    1995-07-01

    The relationship of insect herbivory to conifer physiology is examined. Aspects of nutrient assimilation, nutrient distribution, water stress, and climatic change are correlated to defoliation by insects. Other factors examined include plant age, density, structure, soils, and plant genotype.

  13. Increased expression of matrix metalloproteinases in vivo in scleritis tissue and in vitro in cultured human scleral fibroblasts.

    PubMed Central

    Di Girolamo, N.; Lloyd, A.; McCluskey, P.; Filipic, M.; Wakefield, D.

    1997-01-01

    Scleritis is a sight-threatening inflammatory disorder of the eye characterized by the degradation of scleral matrix. Matrix metalloproteinases (MMPs) are ubiquitous proteolytic enzymes important in physiological and pathological processes, the activity of which is stringently controlled by the action of a family of natural antagonists, the tissue inhibitors of matrix metalloproteinases (TIMPs). We hypothesized that enhanced expression of MMPs, without the negative regulatory influence of TIMPs, may be a key feature of tissue destruction in inflammatory eye diseases, such as scleritis. The aim of this study was to localize and characterize cells expressing MMPs and TIMPs in sclera affected by necrotizing scleritis and, in a parallel study, to establish whether cytokines modulate MMP expression in cultured human scleral fibroblasts. In situ hybridization and immunohistochemical analyses indicated that resident scleral fibroblasts as well as inflammatory cells such as macrophages and T lymphocytes express stromelysin, gelatinase B, and TIMP-1 in necrotizing scleritis tissue. In addition, cytoplasmic immunoreactivity for tumor necrosis factor-alpha, an inducer of MMPs, was detected in infiltrating inflammatory cells. Cultured scleral fibroblasts stimulated with the combination of interleukin-1 alpha plus tumor necrosis factor-alpha increased TIMP-1 mRNA twofold above constitutive levels. By contrast, these cytokines induced a sevenfold increase in the steady-state levels of stromelysin mRNA. Using Western blotting, stromelysin and TIMP-1 protein production paralleled mRNA induction in cytokine-stimulated human scleral fibroblasts. Culture supernatants harvested from cytokine-stimulated human scleral fibroblasts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis gelatin substrate zymography. Our results revealed a prominent 92-kd gelatinolytic band corresponding to gelatinase B, which was inducible with interleukin-1 alpha. These data provide

  14. MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND GLUTATHIONE PEROXIDASE IN CULTURED CELLS AND TISSUE

    PubMed Central

    Weydert, Christine J.; Cullen, Joseph J.

    2010-01-01

    Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, while the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein needed for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissue and cells. In general, these assays require 24 to 48 hours to complete. PMID:20057381

  15. Improving Normal Tissue Complication Probability Models: The Need to Adopt a 'Data-Pooling' Culture

    SciTech Connect

    Deasy, Joseph O.; Bentzen, Soren M.; Jackson, Andrew; Ten Haken, Randall K.; Yorke, Ellen D.; Constine, Louis S.; Sharma, Ashish; Marks, Lawrence B.

    2010-03-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy.

  16. Improving normal tissue complication probability models: the need to adopt a "data-pooling" culture.

    PubMed

    Deasy, Joseph O; Bentzen, Søren M; Jackson, Andrew; Ten Haken, Randall K; Yorke, Ellen D; Constine, Louis S; Sharma, Ashish; Marks, Lawrence B

    2010-03-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy. PMID:20171511

  17. IMPROVING NORMAL TISSUE COMPLICATION PROBABILITY MODELS: THE NEED TO ADOPT A “DATA-POOLING” CULTURE

    PubMed Central

    Deasy, Joseph O.; Bentzen, Søren M.; Jackson, Andrew; Ten Haken, Randall K.; Yorke, Ellen D.; Constine, Louis S.; Sharma, Ashish; Marks, Lawrence B.

    2010-01-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy. PMID:20171511

  18. Development of a complex bone tissue culture system based on cellulose nanowhisker mechanical strain.

    PubMed

    Kim, Dae Seung; Jung, Sang-Myung; Yoon, Gwang Heum; Lee, Hoo Cheol; Shin, Hwa Sung

    2014-11-01

    In bone tissue engineering, scaffolds have been investigated for their ability to support osteoblast growth and differentiation for recovery of damaged bones. Tunicate cellulose nanowhisker (CNW) film and mechanical strain were assessed for their suitability for osteoblasts. In this study, sulfuric acid hydrolysis extraction of tunicates integuments was conducted to obtain CNWs, which were found to be acceptable for adhering, growing, and differentiating osteoblasts without cytotoxicity. Mechanical stress enhanced osteoblast differentiation, and cell survival rate was recovered at around day 3, although there was a slight increase in cell death at day 1 after stimulation. We also found that intracellular flux of calcium ion was related to increased differentiation of CNWs under mechanical stress. Overall, we demonstrated the suitability of tunicate CNWs as a scaffold for bone tissue engineering and developed a complex system based on CNW for osteoblast growth and differentiation that will be useful for bone substitute fabrication. PMID:25454753

  19. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

    PubMed

    Chen, M H; Wang, P J; Maeda, E

    1987-10-01

    The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions. PMID:24248842

  20. Skeletal muscle regeneration via engineered tissue culture over electrospun nanofibrous chitosan/PVA scaffold.

    PubMed

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

    2016-07-01

    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. PMID:26945909

  1. A hydrophobic perfluoropolyether elastomer as a patternable biomaterial for cell culture and tissue engineering.

    PubMed

    Schulte, Vera A; Hu, Yibing; Diez, Mar; Bünger, Daniel; Möller, Martin; Lensen, Marga C

    2010-11-01

    We present a systematic study of a perfluoropolyether (PFPE)-based elastomer as a new biomaterial. Besides its excellent long-term stability and inertness, PFPE can be decorated with topographical surface structures by replica molding. Micrometer-sized pillar structures led to considerably different cell morphology of fibroblasts. Although PFPE is a very hydrophobic material we could show that PFPE substrates allow cell adhesion and spreading of primary human fibroblasts (HDF) very similar to that observed on standard cell culture substrates. Less advanced cell spreading was observed for L929 (murine fibroblast cell line) cells during the first 5 h in culture which was accompanied by retarded recruitment of α(v)β(3)-integrin into focal adhesions (FAs). After 24 h distinct FAs were evident also in L929 cells on PFPE. Furthermore, organization of soluble FN into a fibrillar ECM network was shown for hdF and L929 cells. Based on these results PFPE is believed to be a suitable substrate for several biological applications. On the one hand it is an ideal cell culture substrate for fundamental research of substrate-independent adhesion signaling due to its different characteristics (e.g. wettability, elasticity) compared to glass or TCPS. On the other hand it could be a promising implant material, especially due to its straightforward patternability, which is a tool to direct cell growth and differentiation. PMID:20708794

  2. Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro.

    PubMed

    Bertolo, Alessandro; Thiede, Thomas; Aebli, Niklaus; Baur, Martin; Ferguson, Stephen J; Stoyanov, Jivko V

    2011-04-01

    The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 μg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-β1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1β and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-β1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1β expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue. PMID:21181480

  3. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue.

    PubMed

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  4. Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

    PubMed Central

    Karjalainen, T; Barc, M C; Collignon, A; Trollé, S; Boureau, H; Cotte-Laffitte, J; Bourlioux, P

    1994-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment. Images PMID:7927694

  5. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

    PubMed Central

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V.; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  6. Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

    SciTech Connect

    Kabiri, Azadeh; Esfandiari, Ebrahim; Hashemibeni, Batool; Kazemi, Mohammad; Mardani, Mohammad; Esmaeili, Abolghasem

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer We investigated effects of FGF-2 on hADSCs. Black-Right-Pointing-Pointer We examine changes in the level of gene expressions of SOX-9, aggrecan and collagen type II and type X. Black-Right-Pointing-Pointer FGF-2 induces chondrogenesis in hADSCs, which Bullet Increasing information will decrease quality if hospital costs are very different. Black-Right-Pointing-Pointer The result of this study may be beneficial in cartilage tissue engineering. -- Abstract: Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs) are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.

  7. Gene Expression Profiles of Human Adipose Tissue-Derived Mesenchymal Stem Cells Are Modified by Cell Culture Density

    PubMed Central

    Yoo, Keon Hee; Lee, Tae-Hee; Kim, Hye Jin; Jang, In Keun; Chun, Yong Hoon; Kim, Hyung Joon; Park, Seung Jo; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Sung, Ki Woong; Koo, Hong Hoe

    2014-01-01

    Previous studies conducted cell expansion ex vivo using low initial plating densities for optimal expansion and subsequent differentiation of mesenchymal stem cells (MSCs). However, MSC populations are heterogeneous and culture conditions can affect the characteristics of MSCs. In this study, differences in gene expression profiles of adipose tissue (AT)-derived MSCs were examined after harvesting cells cultured at different densities. AT-MSCs from three different donors were plated at a density of 200 or 5,000 cells/cm2. After 7 days in culture, detailed gene expression profiles were investigated using a DNA chip microarray, and subsequently validated using a reverse transcription polymerase chain reaction (RT-PCR) analysis. Gene expression profiles were influenced primarily by the level of cell confluence at harvest. In MSCs harvested at ∼90% confluence, 177 genes were up-regulated and 102 genes down-regulated relative to cells harvested at ∼50% confluence (P<0.05, FC>2). Proliferation-related genes were highly expressed in MSCs harvested at low density, while genes that were highly expressed in MSCs harvested at high density (∼90% confluent) were linked to immunity and defense, cell communication, signal transduction and cell motility. Several cytokine, chemokine and growth factor genes involved in immunosuppression, migration, and reconstitution of damaged tissues were up-regulated in MSCs harvested at high density compared with MSCs harvested at low density. These results imply that cell density at harvest is a critical factor for modulating the specific gene-expression patterns of heterogeneous MSCs. PMID:24400072

  8. Six Month Report on Tissue Cultured Avian Skeletal Myofibers in the STL/A Module Aboard STS-77

    NASA Technical Reports Server (NTRS)

    Vandenburgh, Herman H.

    1997-01-01

    Space travel is know to effect skeletal muscle, causing rapid and pronounced atrophy in humans and animals, even when strenuous exercise is used as a countermeasure. The cellular and molecular bases of this atrophy are unknown. Space travel may cause muscle atrophy by a direct effect on the muscle fibers and/or indirectly by reducing circulating levels of growth factors such as growth hormone. The recent development of a tissue culture incubator system for Shuttle Middeck basic science experiments [Space Tissue Loss (STL) Module] by the Walter Reed Army Institute of Research (WRAIR) allows the study of the effects of space travel directly on isolated skeletal myofibers. Avian bioartificial skeletal muscle 'organoids' containing differentiated skeletal myofibers and connective tissue fibroblasts were flown aboard the Space Shuttle (Space Transportation System, STS) on Flight STS-77, a repeat of a similar experiment flown on STS-66. The results from these two flight experiments show for the first time that space travel has a direct effect on skeletal muscle cells separate from any systemic effects resulting from altered circulating growth factors.

  9. Endophytic bacteria in plant tissue culture: differences between easy- and difficult-to-propagate Prunus avium genotypes.

    PubMed

    Quambusch, Mona; Pirttilä, Anna Maria; Tejesvi, Mysore V; Winkelmann, Traud; Bartsch, Melanie

    2014-05-01

    The endophytic bacterial communities of six Prunus avium L. genotypes differing in their growth patterns during in vitro propagation were identified by culture-dependent and culture-independent methods. Five morphologically distinct isolates from tissue culture material were identified by 16S rDNA sequence analysis. To detect and analyze the uncultivable fraction of endophytic bacteria, a clone library was established from the amplified 16S rDNA of total plant extract. Bacterial diversity within the clone libraries was analyzed by amplified ribosomal rDNA restriction analysis and by sequencing a clone for each identified operational taxonomic unit. The most abundant bacterial group was Mycobacterium sp., which was identified in the clone libraries of all analyzed Prunus genotypes. Other dominant bacterial genera identified in the easy-to-propagate genotypes were Rhodopseudomonas sp. and Microbacterium sp. Thus, the community structures in the easy- and difficult-to-propagate cherry genotypes differed significantly. The bacterial genera, which were previously reported to have plant growth-promoting effects, were detected only in genotypes with high propagation success, indicating a possible positive impact of these bacteria on in vitro propagation of P. avium, which was proven in an inoculation experiment. PMID:24812040

  10. Acoustic Monitoring of Insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Farmers, grain elevator managers, and food processors often sample grain for insect damaged kernels and numbers of live adult insects but these easily obtained measurements of insect levels do not provide reliable estimates of the typically much larger populations of internally feeding immature inse...

  11. Exploring Sound with Insects

    ERIC Educational Resources Information Center

    Robertson, Laura; Meyer, John R.

    2010-01-01

    Differences in insect morphology and movement during singing provide a fascinating opportunity for students to investigate insects while learning about the characteristics of sound. In the activities described here, students use a free online computer software program to explore the songs of the major singing insects and experiment with making…

  12. Insects and Spiders.

    ERIC Educational Resources Information Center

    National Audubon Society, New York, NY.

    This set of teaching aids consists of nine Audubon Nature Bulletins, providing teachers and students with informational reading on insects and spiders. The bulletins have these titles: What Good Are Insects, How Insects Benefit Man, Life of the Honey Bee, Ants and Their Fascinating Ways, Mosquitoes and Other Flies, Caterpillars, Spiders and Silk,…

  13. Insects and Others.

    ERIC Educational Resources Information Center

    Mills, Richard

    1984-01-01

    Several ideas for observing insects and soil animals in the classroom are provided. Also provided are: (1) procedures for making insect cages with milk cartons; (2) suggestions for collecting and feeding insects; and (3) techniques for collecting and identifying soil animals. (BC)

  14. Interdisciplinary Outdoor Education, Insects.

    ERIC Educational Resources Information Center

    Orsborn, Edward E.

    This manual is a teacher's resource and guide book describing activities for elementary students involving the collecting, killing, preserving, and identification of insects. Most activities relate to collecting and identifying, but activities involving terrariums and hatcheries, finding hidden insects, and insect trapping are also described.…

  15. Sunflower insect pests

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like other annual crops, sunflowers are fed upon by a variety of insect pests capable of reducing yields. Though there are a few insects which are considered consistent or severe (e.g., sunflower moth, banded sunflower moth, red sunflower seed weevil), many more insects are capable of causing proble...

  16. [Establishment of cell culture system of rough-legged buzzard and biological characteristic analysis on different tissue cells cultured in vitro].

    PubMed

    Liu, Gang; Wang, Rong-Rong; Li, Yun-Xia; Dai, Yan-Feng; Li, Xi-He; Li, Yu; Li, Yao

    2013-06-01

    In total, three different tissues from the rough-legged buzzard were obtained by culture and successfully cryopreserved and then recovered. During the subculture process, biological characteristics including as cell morphology, growth curve, cell adhesion rate, and karyotype were analyzed and compared, and overall all three kinds of tissue cells exhibited fibroblast-like growth. Oviduct-derived cells had the strongest adherent ability, followed by lung-derived cells and trachea-derived cells. The doubling times of lung-derived cells, trachea-derived cells, and oviduct-derived cells were 29.91±0.39 h, 33.18±0.21 h, and 30.67±0.28 h, respectively, with population doubling times 3.54±0.01, 4.52±0.02, and 4.38±0.03, respectively. Likewise, we noted the chromosome number of the rough-legged buzzard was 68, within the typical type of ZW. These results may potentially provide material and a basis for further research in the field, with the successful preservation of genetic information of rough-legged buzzard. PMID:23776002

  17. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts

    PubMed Central

    Iskandar, Anita R.; Xiang, Yang; Frentzel, Stefan; Talikka, Marja; Leroy, Patrice; Kuehn, Diana; Guedj, Emmanuel; Martin, Florian; Mathis, Carole; Ivanov, Nikolai V.; Peitsch, Manuel C.; Hoeng, Julia

    2015-01-01

    Organotypic 3D cultures of epithelial cells are grown at the air–liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model. PMID:26085348

  18. Impact Assessment of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures: A Comparison of Mono-Culture and Coculture Model Containing Fibroblasts.

    PubMed

    Iskandar, Anita R; Xiang, Yang; Frentzel, Stefan; Talikka, Marja; Leroy, Patrice; Kuehn, Diana; Guedj, Emmanuel; Martin, Florian; Mathis, Carole; Ivanov, Nikolai V; Peitsch, Manuel C; Hoeng, Julia

    2015-09-01

    Organotypic 3D cultures of epithelial cells are grown at the air-liquid interface (ALI) and resemble the in vivo counterparts. Although the complexity of in vivo cellular responses could be better manifested in coculture models in which additional cell types such as fibroblasts were incorporated, the presence of another cell type could mask the response of the other. This study reports the impact of whole cigarette smoke (CS) exposure on organotypic mono- and coculture models to evaluate the relevancy of organotypic models for toxicological assessment of aerosols. Two organotypic bronchial models were directly exposed to low and high concentrations of CS of the reference research cigarette 3R4F: monoculture of bronchial epithelial cells without fibroblasts (BR) and coculture with fibroblasts (BRF) models. Adenylate kinase (AK)-based cytotoxicity, cytochrome P450 (CYP) 1A1/1B1 activity, tissue histology, and concentrations of secreted mediators into the basolateral media, as well as transcriptomes were evaluated following the CS exposure. The results demonstrated similar impact of CS on the AK-based cytotoxicity, CYP1A1/1B1 activity, and tissue histology in both models. However, a greater number of secreted mediators was identified in the basolateral media of the monoculture than in the coculture models. Furthermore, annotation analysis and network-based systems biology analysis of the transcriptomic profiles indicated a more prominent cellular stress and tissue damage following CS in the monoculture epithelium model without fibroblasts. Finally, our results indicated that an in vivo smoking-induced xenobiotic metabolism response of bronchial epithelial cells was better reflected from the in vitro CS-exposed coculture model. PMID:26085348

  19. InsectBase: a resource for insect genomes and transcriptomes.

    PubMed

    Yin, Chuanlin; Shen, Gengyu; Guo, Dianhao; Wang, Shuping; Ma, Xingzhou; Xiao, Huamei; Liu, Jinding; Zhang, Zan; Liu, Ying; Zhang, Yiqun; Yu, Kaixiang; Huang, Shuiqing; Li, Fei

    2016-01-01

    The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96,925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22,536 pathways of 78 insects, 678,881 untranslated regions (UTR) of 84 insects and 160,905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes. PMID:26578584

  20. InsectBase: a resource for insect genomes and transcriptomes

    PubMed Central

    Yin, Chuanlin; Shen, Gengyu; Guo, Dianhao; Wang, Shuping; Ma, Xingzhou; Xiao, Huamei; Liu, Jinding; Zhang, Zan; Liu, Ying; Zhang, Yiqun; Yu, Kaixiang; Huang, Shuiqing; Li, Fei

    2016-01-01

    The genomes and transcriptomes of hundreds of insects have been sequenced. However, insect community lacks an integrated, up-to-date collection of insect gene data. Here, we introduce the first release of InsectBase, available online at http://www.insect-genome.com. The database encompasses 138 insect genomes, 116 insect transcriptomes, 61 insect gene sets, 36 gene families of 60 insects, 7544 miRNAs of 69 insects, 96 925 piRNAs of Drosophila melanogaster and Chilo suppressalis, 2439 lncRNA of Nilaparvata lugens, 22 536 pathways of 78 insects, 678 881 untranslated regions (UTR) of 84 insects and 160 905 coding sequences (CDS) of 70 insects. This release contains over 12 million sequences and provides search functionality, a BLAST server, GBrowse, insect pathway construction, a Facebook-like network for the insect community (iFacebook), and phylogenetic analysis of selected genes. PMID:26578584

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

    PubMed Central

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

    2011-01-01

    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. PMID:21501697

  2. Fibronectin fragments alter matrix protein synthesis in cartilage tissue cultured in vitro.

    PubMed

    Xie, D; Hui, F; Homandberg, G A

    1993-11-15

    We reported earlier that fibronectin fragments (Fn-f) added to bovine articular cartilage cultured in serum-free culture causes marked protease expression with resultant proteoglycan (PG) degradation and release into the culture media. We have further characterized the effects of Fn-f by studies of the effects on proteoglycan, collagen, general protein, and DNA synthesis and reversibility of the cartilage damage. We report here that the most active Fn-f, a 29-kDa amino-terminal Fn-f, when added to a 1 microM concentration, depressed PG and general protein synthesis in cartilage by over 50% within 24 h, as measured by sulfate and methionine/cysteine incorporation, respectively. This same Fn-f decreased PG synthesis throughout the full thickness cartilage section as shown by autoradiography. PG and general protein synthesis were significantly depressed within 24 h by 29-kDa Fn-f concentrations as low as 10 nM. Synthesis rates were effected by 100-fold lower Fn-f concentrations than was induction of proteinases. Removal of the 29-kDa Fn-f allowed a gain to supernormal levels of PG and protein synthesis. Cartilage damaged to the extent of removal of over 50% of the total PG did not replace PG after over 4 weeks in 10% serum-Dulbecco's modified Eagle minimum with or without added TGF-b1 and rIGF-a. These data show that while the effects of Fn-f on elevating protease expression and depressing PG synthesis are reversible, the resultant cartilage damage is apparently irreversible in vitro. Therefore, if Fn-f-mediated cartilage damage occurs as part of cartilage disease processes, the pathologic effects would be quite significant. PMID:8239647

  3. Effects of epidermal growth factor on neural crest cells in tissue culture

    SciTech Connect

    Erickson, C.A.; Turley, E.A.

    1987-04-01

    Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the /sup 3/H-labeled proteoglycan. Furthermore, EGF stimulates (/sup 3/H)thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.

  4. Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment

    PubMed Central

    van der Kuip, Heiko; Mürdter, Thomas E; Sonnenberg, Maike; McClellan, Monika; Gutzeit, Susanne; Gerteis, Andreas; Simon, Wolfgang; Fritz, Peter; Aulitzky, Walter E

    2006-01-01

    Background Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential. Methods We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay. Results We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices. Conclusion We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue. PMID:16603054

  5. Loss of estrogen receptor beta expression in malignant human prostate cells in primary cultures and in prostate cancer tissues.

    PubMed

    Pasquali, D; Rossi, V; Esposito, D; Abbondanza, C; Puca, G A; Bellastella, A; Sinisi, A A

    2001-05-01

    The aim of this study was to investigate the expression of estrogen receptor (ER) beta and alpha genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERbeta and ERalpha messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ERalpha messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERbeta (C-terminal) antibody, demonstrated ERbeta protein in all NPEC lysates and in one of the six CPECS: ERalpha protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ERalpha N-terminal domain was used. In contrast, ERalpha protein was not detected in two of the six CPEC lysates when ERalpha C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6-8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERbeta gene is expressed together with ERalpha in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECS: Moreover, in some CPECs, the ERalpha gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways. PMID:11344205

  6. Role of O-methyltransferase in the lignification of Douglas-fir cultured tissue

    SciTech Connect

    Monroe, S.H.

    1983-01-01

    O-methyltransferase (OMT) is a key enzyme in the biosynthesis of lignin. This enzyme was isolated and characterized in an effort to understand why Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) callus tissue does not form appreciable amounts of lignin yet does form large amounts of the related flavonoids and tannins. It was shown that the OMT in the callus tissue is a cell wall associated, membrane-bound enzyme, in contrast to that of all reported plant species and to Douglas-fir seedlings, which have either a microsomal or soluble OMT. The effect this had on the OMT kinetic constants was studied. It was found that the callus OMT had much higher K/sub m/ constants for caffeic acid in both the membrane-bound and free forms compared with seedlings. The callus membrane-bound K/sub m/ for caffeic acid is 333 ..mu..M. The callus membrane-free K/sub m/ for caffeic acid is 250 ..mu..M. The seedling K/sub m/ for caffeic acid is 90 ..mu..M.

  7. Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.).

    PubMed

    Jeong, W J; Min, S R; Liu, J R

    1995-07-01

    Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F1 Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and α-naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl(-1) 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl(-1) 2,4-D and 1 mgl(-1) abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron. PMID:24194314

  8. Studies of mineralization in tissue culture: optimal conditions for cartilage calcification

    NASA Technical Reports Server (NTRS)

    Boskey, A. L.; Stiner, D.; Doty, S. B.; Binderman, I.; Leboy, P.

    1992-01-01

    The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

  9. The lazaroid U-83836E improves the survival of rat embryonic mesencephalic tissue stored at 4 degrees C and subsequently used for cultures or intracerebral transplantation.

    PubMed

    Grasbon-Frodl, E M; Nakao, N; Brundin, P

    1996-01-01

    We assessed the effects of addition of the lazaroid U-83836E to a preservation medium on the survival of rat dopamine neurons stored before culturing or intracerebral transplantation. Embryonic ventral mesencephalic tissue was preserved at 4 degrees C for 8 days with or without the addition of 0.3 mu M of U-83836E to a chemically defined "hibernation" medium. Freshly dissected mesencephalic tissue was used in control groups. For culture experiments, the mesencephalic tissue was dissociated and grown in serum-containing medium. Following 24-48 h in vitro, the number of dopamine neurons in cultures derived from tissue hibernated without the lazaroid was 40% of fresh control, compared with 67% of control in cultures prepared from tissue stored in the presence of U-83836E. When mesencephalic tissue was transplanted to the dopamine-depleted striatum of hemiparkinsonian rats following 8 days storage at 4 degrees C in a medium without U-83836E, the mean number of surviving dopamine neurons in the grafts was significantly reduced to 40% of control. In contrast, grafts of tissue which had been hibernated in U-83836E-containing medium contained as many dopamine neurons as transplants of freshly dissected tissue. High yields of surviving grafted dopamine neurons were correlated to a significantly faster onset of functional recovery of amphetamine-induced motor asymmetry. We conclude that the storage period for rat mesencephalic tissue can be prolonged up to 8 days when using lazaroid-supplemented hibernation medium. As lazaroids have undergone clinical safety testing, the application of lazaroids for tissue storage in clinical transplantation trials can be envisaged. PMID:9138743

  10. Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering.

    PubMed

    Chang, Zhengqi; Hou, Tianyong; Xing, Junchao; Wu, Xuehui; Jin, Huiyong; Li, Zhiqiang; Deng, Moyuan; Xie, Zhao; Xu, Jianzhong

    2014-01-01

    To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF. PMID:25329501

  11. Umbilical Cord Wharton’s Jelly Repeated Culture System: A New Device and Method for Obtaining Abundant Mesenchymal Stem Cells for Bone Tissue Engineering

    PubMed Central

    Xing, Junchao; Wu, Xuehui; Jin, Huiyong; Li, Zhiqiang; Deng, Moyuan; Xie, Zhao; Xu, Jianzhong

    2014-01-01

    To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton’s jelly (hUCMSCs) represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC) tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton’s jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15–20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF) not only made full use of the Wharton’s jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF. PMID:25329501

  12. Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

    PubMed

    Aguiar, F L N; Lunardi, F O; Lima, L F; Rocha, R M P; Bruno, J B; Magalhães-Padilha, D M; Cibin, F W S; Rodrigues, A P R; Gastal, M O; Gastal, E L; Figueiredo, J R

    2016-04-01

    This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 μg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 μg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth. PMID:26777561

  13. Insect Barcode Information System

    PubMed Central

    Pratheepa, Maria; Jalali, Sushil Kumar; Arokiaraj, Robinson Silvester; Venkatesan, Thiruvengadam; Nagesh, Mandadi; Panda, Madhusmita; Pattar, Sharath

    2014-01-01

    Insect Barcode Information System called as Insect Barcode Informática (IBIn) is an online database resource developed by the National Bureau of Agriculturally Important Insects, Bangalore. This database provides acquisition, storage, analysis and publication of DNA barcode records of agriculturally important insects, for researchers specifically in India and other countries. It bridges a gap in bioinformatics by integrating molecular, morphological and distribution details of agriculturally important insects. IBIn was developed using PHP/My SQL by using relational database management concept. This database is based on the client– server architecture, where many clients can access data simultaneously. IBIn is freely available on-line and is user-friendly. IBIn allows the registered users to input new information, search and view information related to DNA barcode of agriculturally important insects.This paper provides a current status of insect barcode in India and brief introduction about the database IBIn. Availability http://www.nabg-nbaii.res.in/barcode PMID:24616562

  14. Distributed Pore Chemistry in Porous Organic Polymers in Tissue Culture Flasks

    NASA Technical Reports Server (NTRS)

    Koontz, Steven L. (Inventor)

    1999-01-01

    A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclose. The substrate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphorylcholine groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge, wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic regions, and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

  15. Optical spectral analysis of ultra-weak photon emission from tissue culture and yeast cells

    NASA Astrophysics Data System (ADS)

    Nerudová, Michaela; Červinková, Kateřina; Hašek, Jiří; Cifra, Michal

    2015-01-01

    Optical spectral analysis of the ultra-weak photon emission (UPE) could be utilized for non-invasive diagnostic of state of biological systems and for elucidation of underlying mechanisms of UPE generation. Optical spectra of UPE from differentiated HL-60 cells and yeast cells (Saccharomyces cerevisiae) were investigated. Induced photon emission of neutrophil-like cells and spontaneous photon emission of yeast cells were measured using highly sensitive photomultiplier module Hamamatsu H7360-01 in a thermally regulated light-tight chamber. The respiratory burst of neutrophil-like HL-60 cells was induced with the PMA (phorbol 12-myristate, 13-acetate). PMA activates an assembly of NADPH oxidase, which induces a rapid formation of reactive oxygen species (ROS). Long-pass edge filters (wavelength 350, from 400 to 600 with 25 nm resolution and 650 nm) were used for optical spectral analysis. Propagation of error of indirect measurements and standard deviation were used to assess reliability of the measured spectra. Results indicate that the photon emission from both cell cultures is detectable in the six from eight examined wavelength ranges with different percentage distribution of cell suspensions, particularly 450-475, 475-500, 500-525, 525-550, 550-575 and 575-600 nm. The wavelength range of spectra from 450 to 550 nm coincides with the range of photon emission from triplet excited carbonyls (350-550 nm). The both cells cultures emitted photons in wavelength range from 550 to 600 nm but this range does not correspond with any known emitter. To summarize, we have demonstrated a clear difference in the UPE spectra between two organisms using rigorous methodology and error analysis.

  16. Cytotoxicity of copper complexes of 2-furaldehyde oxime derivatives in murine and human tissue cultured cell lines.

    PubMed

    Hall, I H; Taylor, K; Miller, M C; Dothan, X; Khan, M A; Bouet, F M

    1997-01-01

    The copper complexes of furan oxime derivatives were found to be potent cytotoxic agents in both murine and human tissue cultured cell lines which were either suspended or solid tumors. The ED50 values were frequently improved over the clinically useful antineoplastic agents. These copper complexes of 2-furaldehyde oximes were effective inhibitors of L1210 lymphoid leukemia DNA synthesis followed by RNA synthesis. Purine synthesis regulatory enzyme activities were markedly reduced by the compounds with marginal inhibition of t-RNA polymerase, and nucleoside kinases activities. L1210 DNA topoisomerase II activity was markedly reduced with IC50 values better than the standard VP-16, etoposide. Yet, the copper complexes caused no further protein linked breaks than VP-16 did, but did block phosphorylation activation of the topoisomerase II enzyme. PMID:9252656

  17. A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture.

    PubMed Central

    Hagenbüchle, O; Wellauer, P K

    1992-01-01

    We describe a rapid and general method for isolating DNA-binding proteins in high yield from purified nuclei of animal cells. The method has been tested for the isolation of a series of different DNA-binding activities including those of transcription factors PTF1 and SP1. The rationale consists of first preparing purified nuclei from tissue or cells in culture by centrifugation over sucrose cushions. A synthetic, biotinylated oligonucleotide bearing the binding site for the protein of interest is then added directly to nuclei resuspended in binding buffer. At the end of the binding reaction, nuclei are removed by centrifugation; and protein-DNA complexes present in the postnuclear supernatant are attached to streptavidin-agarose. Two rounds of DNA-affinity chromatography are carried out to yield highly purified preparations of DNA-binding proteins. Images PMID:1641323

  18. Evaluation of Microorganisms Cultured from Injured and Repressed Tissue Regeneration Sites in Endangered Giant Aquatic Ozark Hellbender Salamanders

    PubMed Central

    Nickerson, Cheryl A.; Ott, C. Mark; Castro, Sarah L.; Garcia, Veronica M.; Molina, Thomas C.; Briggler, Jeffrey T.; Pitt, Amber L.; Tavano, Joseph J.; Byram, J. Kelly; Barrila, Jennifer; Nickerson, Max A.

    2011-01-01

    Investigation into the causes underlying the rapid, global amphibian decline provides critical insight into the effects of changing ecosystems. Hypothesized and confirmed links between amphibian declines, disease, and environmental changes are increasingly represented in published literature. However, there are few long-term amphibian studies that include data on population size, abnormality/injury rates, disease, and habitat variables to adequately assess changes through time. We cultured and identified microorganisms isolated from abnormal/injured and repressed tissue regeneration sites of the endangered Ozark Hellbender, Cryptobranchus alleganiensis bishopi, to discover potential causative agents responsible for their significant decline in health and population. This organism and our study site were chosen because the population and habitat of C. a. bishopi have been intensively studied from 1969–2009, and the abnormality/injury rate and apparent lack of regeneration were established. Although many bacterial and fungal isolates recovered were common environmental organisms, several opportunistic pathogens were identified in association with only the injured tissues of C.a. bishopi. Bacterial isolates included Aeromonas hydrophila, a known amphibian pathogen, Granulicetella adiacens, Gordonai terrae, Stenotrophomonas maltophilia, Aerococcus viridans, Streptococcus pneumoniae and a variety of Pseudomonads, including Pseudomonas aeruginosa, P. stutzeri, and P. alcaligenes. Fungal isolates included species in the genera Penicillium, Acremonium, Cladosporium, Curvularia, Fusarium, Streptomycetes, and the Class Hyphomycetes. Many of the opportunistic pathogens identified are known to form biofilms. Lack of isolation of the same organism from all wounds suggests that the etiological agent responsible for the damage to C. a. bishopi may not be a single organism. To our knowledge, this is the first study to profile the external microbial consortia cultured from a

  19. Chondrocytes within osteochondral grafts are more resistant than osteoblasts to tissue culture at 37°C.

    PubMed

    Bastian, Johannes D; Egli, Rainer J; Ganz, Reinhold; Hofstetter, Willy; Leunig, Michael

    2011-01-01

    It is proposed that an ideal osteochondral allograft for cartilage repair consists of a devitalized bone but functional cartilage. The different modes of nutrient supply in vivo for bone (vascular support) and cartilage (diffusion) suggest that a modulation of storage conditions could differentially affect the respective cells, resulting in the proposed allograft. For this purpose, osteochondral tissues from porcine humeral heads were either cultured at 37°C for up to 24 hr or stored at 4°C for 24 hr, the temperature at which osteochondral allografts are routinely stored. Functionality of the cells was assessed by in situ hybridization for transcripts encoding collagen types I and II. At 37°C, a time-dependent significant reduction of the bone surface covered with functional cells was observed with only 5% ± 5% coverage left at 24 hr compared with 41% ± 10% at 0 hr. Similarly, cartilage area containing functional cells was significantly reduced from 84% ± 7% at 0 hr to 70% ± 3% after 24 hr. After 24 hr at 4°C, a significantly reduced amount of functional cells covering bone surfaces was observed (27% ± 5%) but not of cells within the cartilage (79% ± 8%). In the applied experimental setup, bone cells were more affected by tissue culture at 37°C than cartilage cells. Even though chondrocytes appear to be more sensitive to 37°C than to 4°C, the substantially reduced amount of functional bone cells at 37°C warrants further investigation of whether a preincubation of osteochondral allografts at 37°C--prior to regular storage at 4°C--might result in an optimized osteochondral allograft with devitalized bone but viable cartilage. PMID:21275527

  20. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function.

    PubMed

    Ostrovidov, Serge; Ahadian, Samad; Ramon-Azcon, Javier; Hosseini, Vahid; Fujie, Toshinori; Parthiban, S Prakash; Shiku, Hitoshi; Matsue, Tomokazu; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2014-11-13

    Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co-cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf-5, myogenin, Mefc2, MLP), muscle maturation markers (MHC-IId/x, MHC-IIa, MHC-IIb, MHC-pn, α-actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR-ε) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25393357