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Sample records for 24-well tissue culture

  1. Rickettsia rickettsii isolation from naturally infected Amblyomma parvum ticks by centrifugation in a 24-well culture plate technique

    PubMed Central

    Dzul-Rosado, K.; Peniche-Lara, G.; Tello-Martín, R.; Zavala-Velázquez, J.; Pacheco, R. de Campos; Labruna, M.B.; Sánchez, E.C.; Zavala-Castro, J.

    2013-01-01

    Rocky Mountain spotted fever is an acute illness caused by Rickettsia rickettsii (R. rickettsii) and is transmitted by the bite of ticks of the genera Dermacentor, Amblyomma and Rhipicephalus. The illness results in a high mortality rate and may be easily confused with other febrile syndromes. In Yucatan State, Mexico, childhood cases with a high mortality have been reported. In this work we report the isolation of a Mexican R. rickettsii strain from a tick egg mass using an alternative method for Rickettsia isolation with 24-well plates. We also identified a potential vector of R. rickettsii in the southeast of Mexico, which is Amblyomma parvum. PMID:26623321

  2. 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.

  3. 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…

  4. 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.

  5. Tissue culture: the unrealized potential

    PubMed Central

    2007-01-01

    Lack of differentiated functions of the tissue of origin in tissue culture thought to be due to dedifferentiation was shown to be due to selective overgrowth of fibroblasts. Enrichment culture techniques, (alternate animal and culture passage), designed to give the functionally differentiated cells selective advantage over the fibroblasts resulted in a large number of functionally differentiated clonal strains. Thus the dogma of dedifferentiation was destroyed. It is proposed to substitute the dedifferentiation hypothesis with the hypothesis that cells in culture accurately represent cells in vivo without the complex in vivo environment. With the development of hormonally defined media, combined with functionally differentiated clonal cell lines, the potential of tissue culture studies is greatly augmented. Hormonal responses and dependencies can be discovered in culture and the discovery of dependencies of cancer cells has led to a new rationale for therapy. PMID:19003154

  6. History of plant tissue culture.

    PubMed

    Thorpe, Trevor A

    2007-10-01

    Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist, Haberlandt, at the begining of the 20th century. The early studies led to root cultures, embryo cultures, and the first true callus/tissue cultures. The period between the 1940s and the 1960s was marked by the development of new techniques and the improvement of those that were already in use. It was the availability of these techniques that led to the application of tissue culture to five broad areas, namely, cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), plant modification and improvement, pathogen-free plants and germplasm storage, clonal propagation, and product (mainly secondary metabolite) formation, starting in the mid-1960s. The 1990s saw continued expansion in the application of the in vitro technologies to an increasing number of plant species. Cell cultures have remained an important tool in the study of basic areas of plant biology and biochemistry and have assumed major significance in studies in molecular biology and agricultural biotechnology. The historical development of these in vitro technologies and their applications are the focus of this chapter.

  7. History of plant tissue culture.

    PubMed

    Thorpe, Trevor

    2012-01-01

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

  8. Biomedical advances from tissue culture.

    PubMed

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

    2013-12-01

    The demonstration that the "dedifferentiation" of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that cells in culture accurately represent cells in vivo but without the complex in vivo environment. This concept has been strengthened with the development of hormonally defined culture media in combination with functionally differentiated clonal cell lines, which have augmented the potential of tissue culture studies. The use of hormonally defined media in place of serum-supplemented media demonstrates that hormonal responses and dependencies can be discovered in culture. Discoveries of hormonal dependencies of cancer cells has led to therapies targeting intracellular signaling pathways while discoveries of hormonal responses of pluripotent cells are helping to identify the potential application of stem cells. In these and other ways tissue culture technology will continue to contribute to solving problems of human health.

  9. 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.

  10. 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.

  11. 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.

  12. Cell agglomeration in the wells of a 24-well plate using acoustic streaming.

    PubMed

    Kurashina, Yuta; Takemura, Kenjiro; Friend, James

    2017-02-28

    Cell agglomeration is essential both to the success of drug testing and to the development of tissue engineering. Here, a MHz-order acoustic wave is used to generate acoustic streaming in the wells of a 24-well plate to drive particle and cell agglomeration. Acoustic streaming is known to manipulate particles in microfluidic devices, and even provide concentration in sessile droplets, but concentration of particles or cells in individual wells has never been shown, principally due to the drag present along the periphery of the fluid in such a well. The agglomeration time for a range of particle sizes suggests that shear-induced migration plays an important role in the agglomeration process. Particles with a diameter of 45 μm agglomerated into a suspended pellet under exposure to 2.134 MHz acoustic waves at 1.5 W in 30 s. Additionally, BT-474 cells also agglomerated as adherent masses at the center bottom of the wells of tissue-culture treated 24-well plates. By switching to low cell binding 24-well plates, the BT-474 cells formed suspended agglomerations that appeared to be spheroids, fully fifteen times larger than any cell agglomerates without the acoustic streaming. In either case, the viability and proliferation of the cells were maintained despite acoustic irradiation and streaming. Intermittent excitation was effective in avoiding temperature excursions, consuming only 75 mW per well on average, presenting a convenient means to form fully three-dimensional cellular masses potentially useful for tissue, cancer, and drug research.

  13. 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.

  14. 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)

  15. 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

  16. Artemisinin production in Artemisia annua tissue cultures

    SciTech Connect

    Martinez Isaza, B.C.

    1988-01-01

    Production of artemisinin was studied in both plants and tissue cultures of Artemisia annua L. Incorporation of (3{prime}-{sup 14}C) mevalonic acid sodium salt into artemisinin or arteannuin B was not found when field-grown plants were fed once with 10 or 50 {mu}Ci and harvested after 44, 144 or 288 hr. Artemisinin was not present in root organ cultures, but was present in the shoot cultures in a concentration of less than 5 mg/100 g dry weight. The content of artemisinin in a shoot culture line with elongated and indented shoots was significantly higher at p value of 0.01 from that with short and compact shoots. Induction of roots on shoot cultures was associated with increased artemisinin production. Shoot cultures that developed into plants with roots had higher artemisinin content than those shoots cultures with aerial roots, or shoots cultures with basal roots. The artemisinin content in shoot cultures apparently increased with age. Preliminary studies on the metabolism of arteannuin B demonstrated that shoot cultures absorbed the exogenous arteannuin B from the medium without an increase in artemisinin content.

  17. 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.

  18. 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.

  19. 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

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  20. 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

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  1. 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

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  2. 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

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  3. 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

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell...

  4. Lipid Accumulation in Hypoxic Tissue Culture Cells

    PubMed Central

    Gordon, Gerald B.; Barcza, Maureen A.; Bush, Marilyn E.

    1977-01-01

    Lipid droplets have long been recognized by light microscopy to accumulate in hypoxic cells both in vivo and in vitro. In the present tissue culture experiments, correlative electron microscopic observations and lipid analyses were performed to determine the nature and significance of lipid accumulation in hypoxia. Strain L mouse fibroblasts were grown in suspension culture, both aerobically and under severe oxygen restriction obtained by gassing cultures daily with an 8% CO2-92% nitrogen mixture. After 48 hours, hypoxic cells showed an increase in total lipid/protein ratio of 42% over control cells. Most of this increase was accounted for by an elevation in the level of cellular triglyceride from 12.3 ± 0.9 μg/mg cell protein in aerobic cultures to 41.9 ± 0.7 in the hypoxic cultures, an increase of 240%. Levels of cellular free fatty acids (FFA) were 96% higher in the hypoxic cultures. No significant changes in the levels of cellular phospholipid or cholesterol were noted. Electron microscopic examination revealed the accumulation of homogeneous cytoplasmic droplets. The hypoxic changes were reversible upon transferring the cultures to aerobic atmospheres with disappearance of the lipid. These experiments indicate that hypoxic injury initially results in triglyceride and FFA accumulation from an inability to oxidize fatty acids taken up from the media and not from autophagic processes, as described in other types of cell injury associated with the sequestration of membranous residues and intracellular cholesterol and phospholipid accumulation. ImagesFigure 3Figure 4Figure 5Figure 6Figure 7Figure 1Figure 2 PMID:196505

  5. Aeroponics for the culture of organisms, tissues and cells.

    PubMed

    Weathers, P J; Zobel, R W

    1992-01-01

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

  6. 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.

  7. Organotypic slice culture of embryonic brain tissue.

    PubMed

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

    2007-12-01

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

  8. 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).

  9. 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.

  10. A 3D-Printed Oxygen Control Insert for a 24-Well Plate.

    PubMed

    Brennan, Martin D; Rexius-Hall, Megan L; Eddington, David T

    2015-01-01

    3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture.

  11. A 3D-Printed Oxygen Control Insert for a 24-Well Plate

    PubMed Central

    Brennan, Martin D.; Rexius-Hall, Megan L.; Eddington, David T.

    2015-01-01

    3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture. PMID:26360882

  12. 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.

  13. Basic techniques in mammalian cell tissue culture.

    PubMed

    Phelan, Katy; May, Kristin M

    2015-03-02

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.

  14. 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.

  15. Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial. PMID:21326169

  16. Alkaloid production by callous tissue cultures of Cereus peruvianus (Cactaceae).

    PubMed

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

    2003-02-01

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

  17. 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.

  18. 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)

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

    PubMed

    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.

  20. 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.

  1. 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.

  2. [Issues of large scale tissue culture of medicinal plant].

    PubMed

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

    2014-09-01

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

  3. 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.

  4. Use of diathermy for weeding heterogeneous tissue cultures.

    PubMed

    Marks, R M; Penny, R

    1986-06-01

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

  5. Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium.

    PubMed

    Nakanuma, Y; Katayanagi, K; Kawamura, Y; Yoshida, K

    1997-10-01

    Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis.

  6. Characterization of human myoblast cultures for tissue engineering.

    PubMed

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

    2008-01-01

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

  7. Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

    PubMed

    AbouZid, S

    2014-01-01

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

  8. Selection of bacterial wilt-resistant tomato through tissue culture.

    PubMed

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

    1989-06-01

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

  9. 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.

  10. Bioreactors for tissue mass culture: design, characterization, and recent advances.

    PubMed

    Martin, Yves; Vermette, Patrick

    2005-12-01

    This paper reviews reports on three-dimensional mammalian tissue growth in bioreactors and the corresponding mammalian tissue growth requirements. The needs for nutrient and waste removal of several mammalian tissues are reviewed and compared with the environment of many reactors currently in use such as the continuous stirred tank, the hollow fiber, the Couette-Taylor, the airlift, and the rotating-wall reactors developed by NASA. Many studies conclude that oxygen supply appears to be one of the most important factors limiting tissue growth. Various correlations to describe oxygen mass transfer are presented and discussed with the aim to provide some guidance to design, construct, and test reactors for tissue mass culture. To obtain tissue thickness clinically valuable, dimensionless and other types of analysis tend to point out that diffusive transport will have to be matched with an important convection to bring sufficient oxygen molecular flux to the growing cells located within a tissue mass. As learned from solid-state fermentation and hairy root culture, during the growth of large biomass, heterogeneity (i.e., channeling, temperature gradients, non-uniform cell growth, transfer gradients, etc.) can cause some important problems and these should be addressed in tissue engineering as well. Reactors (along with the scaffolds) should be designed to minimize these issues. The role of the uterus, the reactor built by Nature, is examined, and the environment provided to a growing embryo is reported, yielding possible paths for further reactor developments. Finally, the importance of cell seeding methods is also addressed.

  11. 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.

  12. The use of animal tissues alongside human tissue: Cultural and ethical considerations.

    PubMed

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

    2016-01-01

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

  13. 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)

  14. Susceptibility of Tissue Cultures of Canine Origin to Viruses

    DTIC Science & Technology

    1965-08-01

    the viral flora of beagles, it became evident that detailed knowledge of susceptibilities of tissue cultures of canine originto viruses was of prime...host do not necessarily have the capability of growing viruses which in- fect his species. -15- REFERENCES 1. Clapper, W. E. and F. F. Pindak, " Flora of Healthy

  15. Immunologic and tissue culture approaches to the neurobiology of aging.

    PubMed

    Rogers, J; Rovigatti, U

    1988-01-01

    The neurobiology of aging continues to attract scientists and techniques from the more fundamental disciplines, as witness the great strides now being made from molecular genetic approaches to Alzheimer's disease. The present report is a commentary on reviews of immune mechanisms and tissue culture methods applied to investigations of aging and age-related cognitive dysfunction.

  16. Adaptive image segmentation applied to plant reproduction by tissue culture

    NASA Astrophysics Data System (ADS)

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

    1997-04-01

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

  17. [Tissue culture of medicinal plant and abscisic acid].

    PubMed

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

    2013-01-01

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

  18. 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

  19. 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.

  20. The visual assessment of broth cultures for tissue bank samples.

    PubMed

    Varettas, Kerry

    2017-01-05

    The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5-5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.

  1. 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.

  2. 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

  3. 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.

  4. Preventing Photochemistry in Culture Media by Long-Pass Light Filters Alters Growth of Cultured Tissues

    PubMed Central

    Stasinopoulos, Triant C.; Hangarter, Roger P.

    1990-01-01

    Exposure of plant tissue culture media to light from fluorescent bulbs changed the growth regulating properties of the media. The light caused nutrient medium-dependent photosensitized degradation of the phytohormone indole-3-acetic acid and other media components. Photochemical changes in culture media were caused by light from 290 to 450 nanometers and were prevented with a yellow long-pass filter. The use of appropriately filtered light when culturing plant material can eliminate unnecessary variability by stabilizing the culture media composition. PMID:16667626

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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...

  10. 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.

  11. Adipose tissue-organotypic culture system as a promising model for studying adipose tissue biology and regeneration

    PubMed Central

    Uchihashi, Kazuyoshi; Aoki, Shigehisa; Sonoda, Emiko; Yamasaki, Fumio; Piao, Meihua; Ootani, Akifumi; Yonemitsu, Nobuhisa; Sugihara, Hajime

    2009-01-01

    Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. Our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro. PMID:19794899

  12. Tissue Engineered Bone Grafts: Biological Requirements, Tissue Culture and Clinical Relevance

    PubMed Central

    Fröhlich, Mirjam; Grayson, Warren L.; Wan, Leo Q.; Marolt, Darja; Drobnic, Matej; Vunjak-Novakovic, Gordana

    2009-01-01

    The tremendous need for bone tissue in numerous clinical situations and the limited availability of suitable bone grafts are driving the development of tissue engineering approaches to bone repair. In order to engineer viable bone grafts, one needs to understand the mechanisms of native bone development and fracture healing, as these processes should ideally guide the selection of optimal conditions for tissue culture and implantation. Engineered bone grafts have been shown to have capacity for osteogenesis, osteoconduction, osteoinduction and osteointegration - functional connection between the host bone and the graft. Cells from various anatomical sources in conjunction with scaffolds and osteogenic factors have been shown to form bone tissue in vitro. The use of bioreactor systems to culture cells on scaffolds before implantation further improved the quality of the resulting bone grafts. Animal studies confirmed the capability of engineered grafts to form bone and integrate with the host tissues. However, the vascularization of bone remains one of the hurdles that need to be overcome if clinically sized, fully viable bone grafts are to be engineered and implanted. We discuss here the biological guidelines for tissue engineering of bone, the bioreactor cultivation of human mesenchymal stem cells on three-dimensional scaffolds, and the need for vascularization and functional integration of bone grafts following implantation. PMID:19075755

  13. 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.

  14. 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

  15. Addressing the Instability of DNA Nanostructures in Tissue Culture

    PubMed Central

    2015-01-01

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

  16. Addressing the instability of DNA nanostructures in tissue culture.

    PubMed

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

    2014-09-23

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

  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, 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....

  19. 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....

  20. 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....

  1. 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....

  2. 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....

  3. Organotypic tissue culture investigation of homocysteine thiolactone cardiotoxic effect.

    PubMed

    Lopatina, Ekaterina V; Kipenko, A V; Penniyaynen, V A; Pasatetskaya, N A; Djuric, D; Krylov, B V

    2015-06-01

    Homocysteine thiolactone was demonstrated to inhibit the growth of 10-12-day-old chicken embryo cardiac tissue explants at 7 × 10⁻⁹ -1 × 10⁻³ M concentrations in a dose-dependent manner. The maximal cardiotoxic effect of homocysteine thiolactone was detected at 1 × 10⁻³ M, which corresponds to severe hyperhomocysteinemia. The results of experiments on culturing of cardiac tissue explants in the medium containing homocysteine thiolactone (1 × 10⁻³ M) and ouabain at concentrations regulating the signal-transducing (1 × 10⁻¹⁰ M) and pumping (1 × 10⁻⁸ M) functions of Na⁺,K⁺ -ATPase indicate that the cardiotoxic effect of homocysteine thiolactone is supposed to result from inhibition of the Na⁺,K⁺ -ATPase pumping function.

  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. 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.

  6. 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.

  7. 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.

  8. 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#

  9. 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

  10. Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

    PubMed Central

    Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

    2012-01-01

    Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and

  11. 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.

  12. 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.

  13. Selection and Characterization of Sethoxydim- Tolerant Maize Tissue Cultures 1

    PubMed Central

    Parker, William B.; Somers, David A.; Wyse, Donald L.; Keith, Robin A.; Burton, James D.; Gronwald, John W.; Gengenbach, Burle G.

    1990-01-01

    `Black Mexican Sweet' (BMS) maize (Zea mays L.) tissue cultures were selected for tolerance to sethoxydim. Sethoxydim, a cyclohexanedione, and haloxyfop, an aryloxyphenoxypropionate, exert herbicidal activity on most monocots including maize by inhibiting acetyl-coenzyme A carboxylase (ACCase). Selected line B10S grew on medium containing 10 micromolar sethoxydim. Lines B50S and B100S were subsequent selections from B10S that grew on medium containing 50 and 100 micromolar sethoxydim, respectively. Growth rates of BMS, B10S, B50S, and B100S were similar in the absence of herbicide. Herbicide concentrations reducing growth by 50% were 0.6, 4.5, 35, and 26 micromolar sethoxydim and 0.06, 0.5, 5.4, and 1.8 micromolar haloxyfop for BMS, B10S, B50S, and B100S, respectively. Sethoxydim and haloxyfop concentrations that inhibited ACCase by 50% were similar for BMS, B10S, B50S, and B100S. However, ACCase activities were 6.01, 10.7, 16.1, and 11.4 nmol HCO3− incorporated per milligram of protein per minute in extracts of BMS, B10S, B50S, and B100S, respectively, suggesting that increased wild-type ACCase activity conferred herbicide tolerance. Incorporation of [14C]acetate into the nonpolar lipid fraction was higher for B50S than for BMS in the absence of sethoxydim providing further evidence for an increase in ACCase activity in the selected line. In the presence of 5 micromolar sethoxydim, [14C]acetate incorporation by B50S was similar to that for untreated BMS. The levels of a biotin-containing polypeptide (about 220,000 molecular weight), presumably the ACCase subunit, were increased in the tissue cultures that exhibited elevated ACCase activity indicating overproduction of the ACCase enzyme. Images Figure 3 PMID:16667393

  14. 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.

  15. Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles.

    PubMed

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

    2013-05-01

    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

  16. Delivery of recombinant alphavirus into hippocampal slice tissue culture.

    PubMed

    Lundstrom, Kenneth

    2012-08-01

    The alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have shown reduced cytotoxicity and prolonged expression. This protocol describes gene delivery of recombinant alphavirus to hippocampal slice cultures. Organotypic slices are covered by a layer of glial cells that impedes the penetration of viral particles to the neurons. Thus, viral particles should be injected manually into the extracellular space of the tissue.

  17. Tissue-culture cell fractionation. Fractionation of membranes from tissue-culture cells homogenized by glycerol-induced lysis.

    PubMed

    Graham, J M; Sandall, J K

    1979-07-15

    1. The disruption of various types of tissue-culture cells by (a) incubation in solutions of 1.2 M-glycerol and (b) transfer of the glycerol-loaded cells to relatively hypo-osmotic solutions of 0.25 M-sucrose was studied. 2. Bivalent cations (2mM-Mg2+) were generally included to preserve the nuclei, but some cells (polyoma-virus-transformed baby-hamster kidney cells) failed to be disrupted adequately under these conditions. 3. Other cells (mouse-embryo fibroblasts) required additional gentle Dounce homogenization to effect complete cell breakage. 4. Purification of the whole homogenate was carried out by a combination of differential centrifugation and sedimentation or flotation through sucrose gradients. 5. Enzyme analysis showed that plasma-membrane, endoplasmic-reticulum and mitochondrial fractions were obtained in good yield and purity.

  18. Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.

    PubMed

    Jiao, Jian; Meng, Na; Wang, Hong; Zhang, Luo

    2013-12-01

    The purpose of this study was to compare cell growth characteristics, ciliated cell differentiation, and function of human nasal epithelial cells established as explant outgrowth cultures or dissociated tissue cultures. Human nasal mucosa of the uncinate process was obtained by endoscopy and epithelial cell cultures were established by explant outgrowth or dissociated tissue culture methods. Epithelial cell growth characteristics were observed by inverted phase contrast microscopy. Ciliated cell differentiation was detected by β-tubulin IVand ZO-1 immunocytochemistry. Basal and ATP-stimulated ciliary beat frequency (CBF) was measured using a highspeed digital microscopic imaging system. Both the explant and dissociated tissue cultures established as monolayers with tight junctions and differentiated cell composition, with both types of cultures comprising ciliated and non-ciliated epithelial cells. Fibroblasts were also frequently found in explant cultures but rarely seen in dissociated tissue cultures. In both culture systems, the highest ciliated cell density appeared at 7th-10th culture day and declined with time, with the lifespan of ciliated cells ranging from 14 to 21 days. Overall, 10% of the cells in explant cultures and 20% of the cells in the dissociated tissue cultures were ciliated. These two cultures demonstrated similar ciliary beat frequency values at baseline (7.78 ± 1.99 Hz and 7.91 ± 2.52 Hz, respectively) and reacted equivalently following stimulation with 100 μM ATP. The results of this study indicate that both the explant outgrowth and dissociated tissue culture techniques are suitable for growing well-differentiated nasal ciliated and non-ciliated cells, which have growth characteristics and ciliary activity similar to those of nasal epithelial cells in vivo.

  19. 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.

  20. Studies on collagenase from rheumatoid synovium in tissue culture

    PubMed Central

    Evanson, John M.; Jeffrey, John J.; Krane, Stephen M.

    1968-01-01

    Fragments of synovium from patients with rheumatoid arthritis survive in defined tissue culture medium in the absence of added serum and, after 3-4 days, release into the medium enzyme capable of degrading undenatured collagen. Maximal activity is observed at pH 7-9 but the enzyme is inactive at pH 5. At temperatures of 20° and 27°C, collagen molecules in solution are cleaved into 3/4 and 1/4 length fragments with minimal loss of negative optical rotation, but with loss in specific viscosity of approximately 60%. Above 30°C the fragments begin to denature and denaturation is complete at 37°C. If the enzyme is not inhibited at this stage the large fragments are broken down further to polypeptides of low molecular weight. Reconstituted collagen fibrils and native fibers at 37°C are cleaved to the low molecular weight fragments, although the fibrils are resistant to breakdown at lower temperatures (20°-27°C). It is proposed that the production of such an enzyme by inflamed and proliferating rheumatoid synovium may be responsible for some of the destruction of collagenous structures that accompanies rheumatoid arthritis. Images PMID:4302179

  1. Embryogenesis and plant regeneration of Medicago spp. in tissue culture.

    PubMed

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

    1986-02-01

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

  2. Cloning of medicinal plants through tissue culture--a review.

    PubMed

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

    2007-11-01

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

  3. Tissue culture tube contaminant inhibits excitatory synaptic channels.

    PubMed

    Reuhl, T O; Amador, M; Dani, J A

    1990-09-01

    Nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus oocytes. Large, slowly desensitizing macroscopic currents were induced by rapidly infusing an electrolyte solution containing 50 microM ACh in the presence of atropine. The N-methyl-D-aspartate (NMDA) subset of glutamate receptors was studied in CA1 hippocampal neurons. Macroscopic currents were induced by rapidly applying 30 microM NMDA and 10 microM glycine in the presence of picrotoxin, strychnine and tetrodotoxin. Exposing the electrolyte solutions to Falcon brand polypropylene tissue culture tubes (Becton Dickinson Labware) decreased the current through the nAChR channels or through the NMDA receptor channels (1). Purified water shaken in the Falcon brand tubes showed a broad absorbance peak near 200 nm. Before exposing the water to the Falcon tubes, the purified water gave no absorbance signal. The results indicate that a substance released from the Falcon tubes inhibits the currents through nAChR and NMDA receptor channels. Our experiments were with 50-ml Falcon 2098 tubes from lot numbers 72180188 and 80290188 and with 15-ml Falcon 2097 tubes from lot number 83560283. These were the only Falcon tubes we tested.

  4. A novel automated electrochemical ascorbic acid assay in the 24-well microtiter plate format.

    PubMed

    Intarakamhang, Sireerat; Leson, Christian; Schuhmann, Wolfgang; Schulte, Albert

    2011-02-14

    Automatic ascorbic acid (AA) voltammetry was established in 24-well microtiter plates. The assay used a movable assembly of a pencil rod working, an Ag/AgCl reference and a Pt counter electrode with differential pulse voltammetry (DPV) for concentration-dependent current generation. A computer was in command of electrode (z) and microtiter plate (x, y) positioning and timed potentiostat operation. Synchronization of these actions supported sequential approach of all wells and subsequent execution of electrode treatment procedures or AA voltammetry at defined intervals in a measuring cycle. DPV in well solutions offered a linear current/concentration range between 0.1 and 8.0 mM, a sensitivity of about 1 μA mM(-1) AA, and a detection limit of 50 μM. When used with a calibration curve or standard addition, automated voltammetry of samples with added known amounts of AA demonstrated good recovery rates. Also, the assay achieved the accurate determination of the AA content of vitamin C tablets, a fruit juice and an herbal tea extract. Robotic AA voltammetry has the advantage of conveniently handling multiple samples in a single measuring run without the continuous attention of laboratory personnel. It is a good option when the goal is cost-effective AA screening of sample libraries and has potential for applications in health care and the food processing, cosmetic and pharmaceutical industries.

  5. 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.

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

    PubMed

    García-Gareta, Elena

    2014-07-01

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

  7. Comparative study on Allium schoenoprasum cultivated plant and Allium schoenoprasum tissue culture organs antioxidant status.

    PubMed

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

    2011-11-01

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

  8. 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.

  9. Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

    DTIC Science & Technology

    2013-06-01

    compatible bioreactor and that hyperpolarized 13C spectroscopy could be employed to study real-time metabolism of normal and malignant tissues. The...function of prostate tissue slice cultures (TCSs) in an nuclear magnetic resonance (NMR)-compatible, 3-dimensional tissue culture bioreactor , (2) to use...the TSC/NMR bioreactor model to identify hyperpolarized metabolic biomarkers of prostate cancer presence and aggressiveness, and (3) to use the TSC

  10. Inducible Transposition of a Heat-Activated Retrotransposon in Tissue Culture.

    PubMed

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

    2016-12-23

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

  11. 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.

  12. 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.

  13. Gas-permeable membranes and co-culture with fibroblasts enable high-density hepatocyte culture as multilayered liver tissues.

    PubMed

    Evenou, Fanny; Hamon, Morgan; Fujii, Teruo; Takeuchi, Shoji; Sakai, Yasuyuki

    2011-07-01

    To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three-dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell-cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate-based format, enabling high density hepatocyte culture as a stable 3D-multilayer. Multilayered co-cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen-conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co-cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate-based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co-cultures were maintained for at least 1 week; the so-cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate-based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening.

  14. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    PubMed

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

    2015-08-01

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

  15. 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.

  16. 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.

  17. Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

    PubMed

    Akiyama, Hirokazu; Kobayashi, Asako; Ichimura, Masaki; Tone, Hiroshi; Nakatani, Masaru; Inoue, Minoru; Tojo, Arinobu; Kagami, Hideaki

    2015-11-01

    The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

  18. 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)

  19. Variation in bioactive principles of Artemisia amygdalina Decne. in wild and tissue culture regenerants.

    PubMed

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

    2013-05-01

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

  20. An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

    PubMed

    Li, Xingnan; Ootani, Akifumi; Kuo, Calvin

    2016-01-01

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

  1. In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation.

    PubMed

    Shih, Sharon M-H; Doran, Pauline M

    2009-09-10

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

  2. Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells.

    PubMed

    Frame, Fiona M; Pellacani, Davide; Collins, Anne T; Maitland, Norman J

    2016-01-01

    In order to fully explore the biology of a complex solid tumor such as prostate cancer, it is desirable to work with patient tissue. Only by working with cells from a tissue can we take into account patient variability and tumor heterogeneity. Cell lines have long been regarded as the workhorse of cancer research and it could be argued that they are of most use when considered within a panel of cell lines, thus taking into account specified mutations and variations in phenotype between different cell lines. However, often very different results are obtained when comparing cell lines to primary cells cultured from tissue. It stands to reason that cells cultured from patient tissue represents a close-to-patient model that should and does produce clinically relevant data. This chapter aims to illustrate the methods of processing, storing and culturing cells from prostate tissue, with a description of potential uses.

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

    PubMed

    Elson, K M; Fox, N; Tipper, J L; Kirkham, J; Hall, R M; Fisher, J; Ingham, E

    2015-06-30

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

  4. 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.

  5. Surface functionalization of tissue culture polystyrene plates with hydroxyapatite under body fluid conditions and its effect on differentiation behaviors of mesenchymal stem cells.

    PubMed

    Iijima, Kazutoshi; Suzuki, Ryo; Iizuka, Ayako; Ueno-Yokohata, Hitomi; Kiyokawa, Nobutaka; Hashizume, Mineo

    2016-11-01

    The surfaces of polystyrene (PS) cell culture plates were functionalized with hydroxyapatite (HAp) under body fluid conditions utilizing protein adsorption layers and a pretreatment with an alternate soaking process (ASP) using solutions containing calcium and phosphate ions. Adsorption layers of human serum albumin (HSA) formed on the surface of each well of commercial 24-well PS plates by solution processes. CaCl2 and K2HPO4 solutions were alternately added to the wells, the plates were incubated to form the precursors, and this was followed by the addition of simulated body fluid (SBF) and a further incubation for 24h. These treatments resulted in the surfaces of the PS cell culture plates being completely covered with bone-like HAp. The coating of PS plates with HAp promoted the adhesion of mesenchymal stem cells (MSCs) and maintained cell growth that was as fast as that on tissue culture-treated PS (TCPS) plates. Osteogenic differentiation was greater, whereas adipogenic and chondrogenic differentiation was less in the culture on HAp-coated PS plates than in that on TCPS plates. The present method is useful for preparing HAp-coated PS plates at clean benches without the need for any expensive apparatus. HAp coated on PS plates by this method was a bone-like apatite with high bioactivity; therefore, the present HAp-coated PS plates are promising materials for assays of bone-related cells in the bone remodeling process.

  6. Consistent and heritable alterations of DNA methylation are induced by tissue culture in maize.

    PubMed

    Stelpflug, Scott C; Eichten, Steven R; Hermanson, Peter J; Springer, Nathan M; Kaeppler, Shawn M

    2014-09-01

    Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.

  7. 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.

  8. Apollo 12 lunar material: effects on lipid levels of tobacco tissue cultures.

    PubMed

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

    1972-02-11

    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 percent) of an unidentified compound or compounds were found only in control tissues that were 3 to 9 weeks of age.

  9. Environmental carcinogens in human target tissues in culture. Progress report

    SciTech Connect

    Hsu, I.C.

    1984-10-19

    The metabolism and mutagenicity of 1-nitropyrene, 1-aminopyrene, 2-aminofluorene and acetylaminofluarene in hepatic cell cultures is investigated. Metabolic products were assayed by thin-layer chromatography. Ancillary studies on Ames' assary mutagencity screening, unscheduled DNA synthesis by hepatocytes and binding to nuclear structures are also reported. 1 fig., 1 tab.

  10. 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...

  11. Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices.

    PubMed

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

    1998-01-05

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

  12. Cytokinin Autonomy in Tissue Cultures of Phaseolus: A Genotype-Specific and Heritable Trait

    PubMed Central

    Mok, Machteld C.; Mok, David W. S.; Armstrong, Donald J.; Rabakoarihanta, Aimée; Kim, Sang-Gu

    1980-01-01

    Intra- and interspecific differences in cytokinin requirement were detected in callus cultures of Phaseolus vulgaris L. and P. lunatus L. Of the ten genotypes of P. vulgaris tested in the present study, one required cytokinin for callus growth, six exhibited some to moderate growth on cytokinin-free medium, and the remaining three grew uniformly in the absence of cytokinin. In contrast, six of the P. lunatus genotypes were strictly cytokinin-dependent, while four genotypes displayed irregular amount of callus growth on cytokinin-free medium. The genotype-specific behavior of Phaseolus callus tissues was independent of the tissue of origin and the time in culture. The inheritance of the cytokinin requirement of Phaseolus tissue cultures was studied in hybrid tissues resulting from crosses between a strictly cytokinin-dependent genotype (P.I. 200960) and two independent genotypes (cv. G 50 and P.I. 286303) of P. vulgaris. Fresh weights of hybrid tissues on cytokinin-free medium were intermediate between and significantly different from the parental tissues. No differences were found between reciprocal hybrids. These results suggest that cytokinin autonomy in tissue cultures of P. vulgaris is a genetic trait under nuclear control. Both parental and intermediate phenotypes were recovered in the F2 progeny. The frequency distribution of cytokinin-dependent progeny in F2 and backcross populations indicates that the cytokinin requirement of P. vulgaris callus tissue may be regulated by one set of alleles. PMID:17249014

  13. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  14. Pattern matching and adaptive image segmentation applied to plant reproduction by tissue culture

    NASA Astrophysics Data System (ADS)

    Vazquez Rueda, Martin G.; Hahn, Federico

    1999-03-01

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

  15. Spontaneous aneuploidy and clone formation in adipose tissue stem cells during different periods of culturing.

    PubMed

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

    2009-07-01

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

  16. 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.

  17. 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

  18. Effects of melatonin on ionic currents in cultured ocular tissues.

    PubMed

    Rich, A; Farrugia, G; Rae, J L

    1999-04-01

    The effects of melatonin on ionic conductances in a cultured mouse lens epithelial cell line (alpha-TN4) and in cultured human trabecular meshwork (HTM) cells were measured using the amphotericin perforated patch whole cell voltage-clamp technique. Melatonin stimulated a voltage-dependent Na+-selective current in lens epithelial cells and trabecular meshwork cells. The effects of melatonin were observed at 50 pM and were maximal at 100 microM. Melatonin enhanced activation and inactivation kinetics, but no change was observed in the voltage dependence of activation. The results are consistent with an increase in the total number of ion channels available for activation by membrane depolarization. Melatonin was also found to stimulate a K+-selective current at high doses (1 mM). Melatonin did not affect the inwardly rectifying K+ current or the delayed rectifier type K+ current that has been described in cultured mouse lens epithelial cells. The results show that melatonin specifically stimulated the TTX-insensitive voltage-dependent Na+ current by an apparently novel mechanism.

  19. Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

    PubMed

    Varettas, Kerry

    2013-12-01

    Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

  20. Application of plant tissue cultures in phytoremediation research: incentives and limitations.

    PubMed

    Doran, Pauline M

    2009-05-01

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

  1. Environmental carcinogens in human target tissues in culture. Progress report

    SciTech Connect

    Hsu, I.C.

    1986-02-19

    Cells from different organ or animal species have shown diverse activities in activation and detoxification of chemical carcinogens. Based on the mutation assays, human hepatocytes were more effective than animal hepatocytes in detoxification of aromatic nitrogen compounds. The adduct formation was also different in human and rodent hepatocytes exposed to aminofluorene (AF) or acetylaminofluorene (AAF). Both AF and AAF adduct DNA were observed in rat liver cells exposed to AF or AAF. However, very little acetylation or deacetyl of the DNA adducts occurred in the human hepatocytes. Human hepatocytes treated with AF in primary culture produced mainly AF adducted DNA while AAF treated cells formed AAF adduct DNA. 2 figs., 1 tab.

  2. Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

    PubMed

    Cassells, Alan C

    2012-01-01

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

  3. Molecular Identification of Zygomycetes from Culture and Experimentally Infected Tissues

    PubMed Central

    Schwarz, Patrick; Bretagne, Stéphane; Gantier, Jean-Charles; Garcia-Hermoso, Dea; Lortholary, Olivier; Dromer, Françoise; Dannaoui, Eric

    2006-01-01

    Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues. PMID:16455881

  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.

  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. Clonal propagation of Leptospermum spp. by tissue culture.

    PubMed

    Shipton, W A; Jackes, B R

    1986-02-01

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

  7. Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

    PubMed

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

    2014-04-01

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

  8. 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

  9. 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.

  10. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

    PubMed

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

    2011-01-01

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

  11. 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.

  12. 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

  13. Evaluation of VP22 spread in tissue culture.

    PubMed

    Brewis, N; Phelan, A; Webb, J; Drew, J; Elliott, G; O'Hare, P

    2000-01-01

    We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.

  14. A physiological study of chick myotubes grown in tissue culture

    PubMed Central

    Harris, J. B.; Marshall, M. W.; Wilson, P.

    1973-01-01

    1. A study has been made of some passive and active membrane properties of myotubes of different ages obtained in culture from explants of chick embryo thigh muscle. 2. After 3 days in vitro the mean values for the myotube resting membrane potential and input resistance were - 63·8 mV and 1·30 MΩ respectively. By 13 days these values had fallen to - 51·0 mV and 0·80 MΩ. 3. Current/voltage relations were measured in the presence of tetrodotoxin. The relations were linear for membrane potentials between - 120 and - 35 mV. Further depolarization usually resulted in a delayed increase in conductance which inactivated with time. 4. All myotubes tested using anodal break excitation were capable of generating action potentials. Action potentials were blocked by tetrodotoxin, saxitoxin and procaine. 5. All myotubes were sensitive to iontophoretically applied ACh. The potential change produced by ACh reversed polarity at a membrane potential between 0 and + 10 mV. The depolarization produced by ACh was unaffected by anticholinesterases. 6. The ACh response was blocked by cobra neurotoxin, D-tubocurarine and atropine. 7. The electrical properties of the myotubes appear to resemble those of normal adult twitch-type skeletal muscle fibres. 8. The pharmacological properties of the myotube cholinergic receptor have been compared with those of the neuromuscular junction and the denervated muscle fibre membrane. ImagesPlate 1Plate 2 PMID:4735059

  15. 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.

  16. Plants regenerated from tissue culture contain stable epigenome changes in rice.

    PubMed

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

    2013-03-19

    Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001.

  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.

  18. Aluminum ions stimulate mitosis in murine cells in tissue culture.

    PubMed

    Jones, T R; Antonetti, D L; Reid, T W

    1986-01-01

    Addition of aluminum to the culture medium of Nakano mouse lens epithelial (NMLE) cells and Swiss 3T3K cells induced both 3H-thymidine incorporation and mitosis. This is in contrast to other metal ions such as vanadium, which, at concentrations high enough to increase 3H-thymidine incorporation, actually inhibits mitosis (Jones and Reid, J Cell Physiol 121:199, 1984). Aluminum concentrations between 20 microM and 50 microM were most effective. The 3T3 cells respond to aluminum with a 7.6-fold increase, and NMLE cells respond with a 21-fold increase in 3H-thymidine incorporation. DNA synthesis in NMLE cells was also found to be synergistically stimulated by aluminum and low concentrations of insulin (4.5 X 10(-8) M). A 3.25-hr incubation with 50 microM aluminum was sufficient to induce 50% of maximum 3H-thymidine incorporation during the 40-hr assay. Aluminum-stimulated 3H-thymidine incorporation is inhibited by hydroxyurea, and aluminum causes an increase in cell number. Also, by sedimentation equilibrium analysis of the product of aluminum-stimulated DNA synthesis it was found that a single copy of DNA was synthesized following addition of aluminum to quiescent cells. These facts indicate that aluminum induces both S-phase DNA synthesis and mitosis. However, only 48% of the NMLE cells found to be labeled with DNA went on to divide. In contrast, although only a small percentage of 3T3 cells were found to be labeled after aluminum treatment, all of these cells appeared to go through mitosis.

  19. Fractals in tissue engineering: toward biomimetic cell-culture matrices, microsystems and microstructured implants.

    PubMed

    Díaz Lantada, Andrés; Pareja Sánchez, Beatriz; Gómez Murillo, Cristina; Urbieta Sotillo, Javier

    2013-09-01

    Tissue engineering is a rapidly evolving field in which the complexity of biomaterials and biostructures, with typically non-Euclidean or fractal-like geometries, has to be adequately taken into account for the promotion of enhanced and even personalized diagnostic and therapeutic solutions. This study covers the main applications of fractals in the field of tissue engineering, including their advantages for modeling biological processes and cell-culture procedures, but specially focusing on their benefits for describing the complex geometries and structures of biomaterials (both natural and synthetic), many of which have potential uses for the development of cell culture microsystems, scaffolds for tissue repair and implants for tissue repair in general. We also explore the main supporting design, simulation and manufacturing technologies, as well as the most remarkable difficulties and limitations linked to the generalized use of fractals in engineering design, and also detail some current solution proposals and future directions.

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

    PubMed Central

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

    2015-01-01

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

  1. 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

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

    PubMed

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

    2015-01-01

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

  3. Development of an experimental tissue culture vaccine against Mediterranean theileriosis in Spain.

    PubMed

    Viseras, J; García-Fernández, P; Adroher, F J

    1998-02-01

    Vaccines against Mediterranean theileriosis have been developed in several countries where the disease is an economic problem. Tissue culture vaccines have been widely and successfully used to immunize cattle. Although Mediterranean theileriosis represents a constraint to dairy cattle production in Spain, no vaccines against this disease have been developed previously. The successful development of a tissue culture vaccine consisting of attenuated Theileria annulata schizont infected cells from an enzootic area of Spain and its efficacy under experimental conditions is reported. Vaccinated calves were resistant to homologous challenge showing no signs of theileriosis while non-vaccinated calves showed typical signs of disease.

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

    PubMed Central

    Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-01-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level. PMID:20680682

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

    PubMed

    Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

    2009-03-01

    Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

  10. 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.

  11. 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.}

  12. In vitro development of primordial follicles after long-term culture of goat ovarian tissue.

    PubMed

    Matos, M H T; Bruno, J B; Rocha, R M P; Lima-Verde, I B; Santos, K D B; Saraiva, M V A; Silva, J R V; Martins, F S; Chaves, R N; Báo, S N; Figueiredo, J R

    2011-06-01

    This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH+FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.

  13. 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

  14. 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...

  15. 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.

  16. Image segmentation using common techniques and illumination applied to tissue culture

    NASA Astrophysics Data System (ADS)

    Vazquez Rueda, Martin G.; Hahn, Federico

    1998-03-01

    This paper present the comparation and performance on no adaptive image segmentation techniques using illumination and adaptive image segmentation techniques. Results obtained on indoor plant reproduction by tissue culture, show the improve in time process, simplify the image segmentation process, experimental results are presented using common techniques in image processing and illumination, contrasted with adaptive image segmentation.

  17. 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...

  18. Cell culture in autologous fibrin scaffolds for applications in tissue engineering.

    PubMed

    de la Puente, Pilar; Ludeña, Dolores

    2014-03-10

    In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth. With this review our aim is to follow methods of development, analyze the commercial and autologous fibrins available and assess the possible applications of cell culture in tissue engineering in these three-dimensional structures.

  19. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    PubMed

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

    1993-08-01

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

  20. Culture of domestic cat ovarian tissue in vitro and in the chick embryo chorioallantoic membrane.

    PubMed

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

    2016-10-15

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

  1. 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 ...

  2. Hydrophobic metabolites of 2,4-dichlorophenoxyacetic acid (2,4-D) in cultured coconut tissue.

    PubMed

    López-Villalobos, Arturo; Hornung, Roland; Dodds, Peter F

    2004-10-01

    Cultures of inflorescence and plumular tissues of coconut palm (Cocos nucifera L.) were maintained in the presence of the auxin, [14C]2,4-dichlorophenoxyacetic acid (2,4-D), so that its metabolic fate could be studied. Thin layer chromatography of methanol extracts of the plumular tissue showed that four classes of metabolites, as well as the unchanged acid, were recovered in the extract. In inflorescence tissue, only the unchanged acid and the most polar class of metabolites (metabolite I) were recovered. Metabolite I was shown to consist mostly of a mixture of sugar conjugates and metabolite II (the next most polar) was an unidentified basic metabolite. Metabolites III and IV were both novel triacylglycerol analogues in which one of the natural fatty acids was replaced with a chain-elongated form of 2,4-D. Reversed-phase thin layer chromatography was used to identify the 2,4-D-derived acids and it was found that metabolite III contained the 2,4-dichlorophenoxy-moiety attached to a chain-length of between 2 and 12 carbons, whereas metabolite IV contained 12, 14 and 16 carbon chain lengths. In inflorescence tissue, and in plumular tissue at low sucrose or 2,4-D concentrations and after short periods in culture, metabolite I predominated. The other metabolites increased as a percentage when plumular culture was prolonged or when sucrose or 2,4-D concentrations were raised. These changes correlated with better development of the explant.

  3. Primary Culture of Mycobacterium ulcerans from Human Tissue Specimens after Storage in Semisolid Transport Medium▿

    PubMed Central

    Eddyani, Miriam; Debacker, Martine; Martin, Anandi; Aguiar, Julia; Johnson, Christian R.; Uwizeye, Cécile; Fissette, Krista; Portaels, Françoise

    2008-01-01

    Tissue specimens collected from patients with clinically suspected Buruli ulcer treated in two Buruli ulcer treatment centers in Benin between 1998 and 2004 were placed in semisolid transport medium and transported at ambient temperature for microbiological analysis at the Institute of Tropical Medicine in Antwerp, Belgium. The impact of the delay before microbiological analysis on primary culture of Mycobacterium ulcerans was investigated. The length of storage in semisolid transport medium varied from 6 days to 26 weeks. Of the 1,273 tissue fragments positive for M. ulcerans DNA by an IS2404-specific PCR, 576 (45.2%) yielded positive culture results. The sensitivity of direct smear examination was 64.6% (822/1,273 tissue fragments). The median time required to obtain a positive culture result was 11 weeks. Positive cultures were obtained even from samples kept for more than 2 months at ambient temperatures. Moreover, there was no reduction in the viability of M. ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field. PMID:17989199

  4. 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

    2015-12-08

    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.

  5. 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

  6. 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.

  7. 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.

  8. Studies on the polyphenol metabolism of tissue cultures derived from the tea plant (Camellia sinensis L.)

    PubMed Central

    Forrest, G. I.

    1969-01-01

    1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation. PMID:5821008

  9. 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

  10. Assay of anticancer drugs in tissue culture: cell cultures of biopsies from human astrocytoma.

    PubMed

    Morgan, D; Freshney, R I; Darling, J L; Thomas, D G; Celik, F

    1983-02-01

    A method has been developed for measuring the drug sensitivity of human gliomas in short-term culture, using scintillation counting or autofluorography. Cell cultures prepared from malignant astrocytomas were treated with anticancer drugs whilst in exponential growth in microtitration plates. After drug treatment and a recovery period, residual viability was measured by [3H] leucine incorporation followed by scintillation counting or by [35S] methionine incorporation and autofluorography in situ. In 5 glioma cell lines tested against 6 drugs, the microtitration method correlated well with monolayer cloning. Although replicate samples of the same tumour showed little variation in chemosensitivity, there was marked variation between the chemosensitivities of cultures derived from the tumours of different patients. However, as variability between replicates was apparent during drug exposure or shortly after, it is important to allow the assay to run as long as possible after drug removal. It is hoped that this assay may provide the basis of a method for the prediction of in vivo chemosensitivity or the screening of potential chemotherapeutic drugs.

  11. Assay of anticancer drugs in tissue culture: cell cultures of biopsies from human astrocytoma.

    PubMed Central

    Morgan, D.; Freshney, R. I.; Darling, J. L.; Thomas, D. G.; Celik, F.

    1983-01-01

    A method has been developed for measuring the drug sensitivity of human gliomas in short-term culture, using scintillation counting or autofluorography. Cell cultures prepared from malignant astrocytomas were treated with anticancer drugs whilst in exponential growth in microtitration plates. After drug treatment and a recovery period, residual viability was measured by [3H] leucine incorporation followed by scintillation counting or by [35S] methionine incorporation and autofluorography in situ. In 5 glioma cell lines tested against 6 drugs, the microtitration method correlated well with monolayer cloning. Although replicate samples of the same tumour showed little variation in chemosensitivity, there was marked variation between the chemosensitivities of cultures derived from the tumours of different patients. However, as variability between replicates was apparent during drug exposure or shortly after, it is important to allow the assay to run as long as possible after drug removal. It is hoped that this assay may provide the basis of a method for the prediction of in vivo chemosensitivity or the screening of potential chemotherapeutic drugs. PMID:6297528

  12. 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.

  13. BIOCHEMICAL STUDY OF CELLULAR ANTIGEN-ANTIBODY REACTION IN TISSUE CULTURE

    PubMed Central

    Hayashi, Hideo; Tokuda, Akira; Udaka, Keiji

    1960-01-01

    The correlation between morphological and biochemical changes produced by the antigen-antibody reaction was studied in cultures of tissue monocytes taken from sensitized animals. The cells were grown under conditions which allowed collection of samples from the culture fluid as well as microscopic observation. Introduction of the antigen into the culture medium causes rapid release of a protease characterized by its susceptibility to sulfhydryl block and its optimum pH in the neutral range. Protease activation occurs simultaneously with morphological changes in the cytoplasm of the cultured cells. Delayed changes affecting the mitochondria and Golgi bodies appear after the peak of the proteolytic reaction and may be secondary to it. The gradual inactivation of the protease observed in the course of the antigen-antibody reaction will be discussed in a separate paper. PMID:13712446

  14. Desmoplastic small round cell tumor (DSRCT) xenografts and tissue culture lines: Establishment and initial characterization

    PubMed Central

    MARKIDES, CONSTANTINE S.A.; COIL, DOUGLAS R.; LUONG, LINH H.; MENDOZA, JOHN; KOZIELSKI, TONY; VARDEMAN, DANA; GIOVANELLA, BEPPINO C.

    2013-01-01

    Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm, which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, its younger and overall healthier patient population (compared with other tumor types) and the fact that it lacks definitive histological and immunohistological features, the diagnosis of DSRCT may be frequently delayed or the tumor may be entirely misdiagnosed as a different type of abdominal sarcoma. The present study aimed to rectify the lack of models that exist for this rare neoplasm, through the development of several DSRCT tissue cultures and xenograft lines. Samples were received from surgeries and biopsies from patients worldwide and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected into the dorsal flanks of nude mice. Of the 14 samples received, nine were established into xenograft lines and five into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and demonstrated two different growth rates among the established xenograft lines. Overall, the establishment of these xenograft and tissue culture lines provides researchers with tools to evaluate DSRCT responses to chemotherapy and to investigate DSRCT-specific signaling pathways or mechanisms. PMID:23759995

  15. Growth of Leptospira pomona and Its Effect on Various Tissue Culture Systems1

    PubMed Central

    Miller, Robert E.; Miller, Norman G.; White, Roberta J.

    1966-01-01

    Miller, Robert E. (University of Nebraska College of Medicine, Omaha), Norman G. Miller, and Roberta J. White. Growth of Leptospira pomona and its effect on various tissue culture systems. J. Bacteriol. 92:502–509. 1966.—Leptospira pomona strain 3341 was grown in association with primary fetal bovine kidney (PBK) and human embryonic skin-muscle fibroblastic (HE) cells in Eagle's minimal essential medium (MEM) with 5% sheep serum. Growth curves of leptospires in PBK and HE cell cultures showed no substantial increase in growth above that obtained in Eagle's MEM in the absence of tissue culture cells. This suggested that no stimulatory growth factors for leptospires were produced by the tissue cells. Fibroblastic cells of the PBK monolayer showed separation, deterioration, and, finally, complete disintegration. Epithelial-like cells remained unaffected. HE cells showed the same cytopathic effect as PBK fibroblastic cells, indicating that this effect was not limited to PBK fibroblastic cells. Warthin-Starry stains of PBK and HE cell monolayers showed masses of leptospires adhering to fibroblastic cells, whereas only a few were seen on epithelial-like cells. Large numbers of leptospires on the surface of fibroblastic cells are very likely associated with the cytopathic effect. Dislodgment of leptospires from fibroblastic cells did not increase the total number of spirochetes in the culture. This indicated that leptospiral growth did not occur on the surface of these cells. Images PMID:16562141

  16. 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.

  17. Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata.

    PubMed

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

    2013-10-01

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

  18. 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…

  19. Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.

    PubMed

    Hendijani, Fatemeh

    2017-04-01

    Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits.

  20. Electrospinning adipose tissue-derived extracellular matrix for adipose stem cell culture.

    PubMed

    Francis, Michael P; Sachs, Patrick C; Madurantakam, Parthasarathy A; Sell, Scott A; Elmore, Lynne W; Bowlin, Gary L; Holt, Shawn E

    2012-07-01

    Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.

  1. Evaluation of Biocompatibility of Alloplastic Materials: Development of a Tissue Culture In Vitro Test System.

    PubMed

    Gerullis, Holger; Georgas, Evangelos; Eimer, Christoph; Goretzki, Peter E; Lammers, Bernhard J; Klosterhalfen, Bernd; Boros, Mihaly; Wishahi, Mohamed; Heusch, Gerd; Otto, Thomas

    2011-12-01

    Optimized biocompatibility is a major requirement for alloplastic materials currently applied in surgical approaches for hernia, incontinence, and prolapse situations. Tissue ingrowth/adherence and formation of connective tissue seem to have important influence in mesh incorporation at the implant site. In an in vitro approach we randomly investigated 7 different mesh types currently used in surgeries with various indications with regard to their adherence performance. Using a tissue culture approach, meshes were incubated with tissue representative of fibroblasts, muscle cells, and endothelial cells originating from 10 different patients. After 6 weeks, the meshes were assessed microscopically and a ranking of their adherence performance was established. Tissue culture was successful in 100% of the probes. We did not remark on interindividual differences concerning the growth and adherence performance after incubation with the different meshes in the investigated 10 patients. The ranking was consistent in all patients. In this test system, PVDF Dynamesh® (FEG Textiltechnik, Aachen, Germany) was the mesh with the best growth-in score. The test system was feasible and reproducible. Pore size seems to be a predictor of adherence performance. The test system may be a helpful tool for further investigations, and the predictive value should be assessed in further in vitro and in vivo experiments.

  2. Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

    NASA Astrophysics Data System (ADS)

    Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

    2017-01-01

    Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

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

    PubMed

    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.

  4. 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

  5. Primary liver cells cultured on carbon nanotube substrates for liver tissue engineering and drug discovery applications.

    PubMed

    Che Abdullah, Che Azurahanim; Azad, Chihye Lewis; Ovalle-Robles, Raquel; Fang, Shaoli; Lima, Marcio D; Lepró, Xavier; Collins, Steve; Baughman, Ray H; Dalton, Alan B; Plant, Nick J; Sear, Richard P

    2014-07-09

    Here, we explore the use of two- and three-dimensional scaffolds of multiwalled-carbon nanotubes (MWNTs) for hepatocyte cell culture. Our objective is to study the use of these scaffolds in liver tissue engineering and drug discovery. In our experiments, primary rat hepatocytes, the parenchymal (main functional) cell type in the liver, were cultured on aligned nanogrooved MWNT sheets, MWNT yarns, or standard 2-dimensional culture conditions as a control. We find comparable cell viability between all three culture conditions but enhanced production of the hepatocyte-specific marker albumin for cells cultured on MWNTs. The basal activity of two clinically relevant cytochrome P450 enzymes, CYP1A2 and CYP3A4, are similar on all substrates, but we find enhanced induction of CYP1A2 for cells on the MWNT sheets. Our data thus supports the use of these substrates for applications including tissue engineering and enhancing liver-specific functions, as well as in in vitro model systems with enhanced predictive capability in drug discovery and development.

  6. Plant tissue culture--an opportunity for the production of nutraceuticals.

    PubMed

    Lucchesini, Mariella; Mensuali-Sodi, Anna

    2010-01-01

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

  7. 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

  8. [Identification of fungi in tissues and cultures: the importance of argirophilic substances on its cellular walls].

    PubMed

    Piva, J R; Ortega, H H; Canal, A M; Piva, C E; Reus, V; Seib, E P

    2001-01-01

    An experimental development based on the combination of microwaves action with one of the methods of silver staining by Del Río Hortega is presented. Material from pathological tissues and culture of fungi were studied. Besides morphological studies, were considered the causes of reduction from ionic to metalic silver, some characteristics of the silver reagent and its relationship with histochemical constitution of cellular walls. It is pointed the rapidity in fungi demonstration, the satisfactory definition of affected tissues, the advantages of working with a stable reagent, the omission of carcinogenetic substances, the possibility of stain fungical structures in previously stained materials with anilinic techniques, and the extent of the method to cultured materials without necessity of previous formaldehidic fixation.

  9. Development of tissue culture techniques and hardware to study mineralization under microgravity conditions

    NASA Astrophysics Data System (ADS)

    van Loon, J. J. W. A.; Veldhuijzen, J. P.; Windgassen, E. J.; Brouwer, T.; Wattel, K.; van Vilsteren, M.; Maas, P.

    1994-08-01

    To study the effects of weightlessness on mouse fetal long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with Na-β-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type I experiment containers. The containers can be flushed with an air/5% CO2 gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of μ-gravity. Using 16 day old metatarsals the in vitro calcification process under μ-gravity conditions can be studied for a 4 day period.

  10. Effect of Endophytic Fusarium oxysporum on Host Preference of Radopholus similis to Tissue Culture Banana Plants.

    PubMed

    Athman, Shahasi Y; Dubois, Thomas; Coyne, Daniel; Gold, Clifford S; Labuschagne, Nico; Viljoen, Altus

    2006-12-01

    The burrowing nematode Radopholus similis is one of the major constraints to banana (Musa spp.) production worldwide. Resource-poor farmers can potentially manage R. similis by using naturally occurring banana endophytes, such as nonpathogenic Fusarium oxysporum, that are inoculated into tissue culture banana plantlets. At present, it is unclear at what stage in the R. similis infection process the endophytes are most effective. In this study, the effect of three endophytic F. oxysporum isolates (V5w2, Eny1.31i and Eny7.11o) on R. similis host preference of either endophyte-treated or untreated banana plants was investigated. No differences were observed between the proportion of nematodes attracted to either root segments excised from endophyte-treated or untreated plants, or in experiments using endophyte-treated and untreated tissue culture banana plantlets. These results imply that the early processes of banana plant host recognition by R. similis are not affected by endophyte infection.

  11. Biochemical Basis of Resistance of Tobacco Callus Tissue Cultures to Hydroxyphenylethylamines.

    PubMed Central

    Negrel, J.; Javelle, F.; Paynot, M.

    1993-01-01

    It has been reported that hydroxyphenylethylamines, such as tyramine and octopamine, are toxic to tobacco (Nicotiana tabacum L.) callus cultures grown in the presence of auxins, whereas calli grown in the presence of cytokinins and crown gall cultures are resistant to these amines (P. Christou and K.A. Barton [1989] Plant Physiol 89: 564-568). In an attempt to understand the underlying mechanism of this resistance, we compared the fates of tyramine in tyramine-sensitive and tyramine-resistant tobacco tissue cultures (cv Xanthi nc). The very rapid formation of black-colored oxidation products from tyramine in sensitive tissues suggested that the toxicity might be caused by the oxidation of tyramine by phenol oxidases present in the tissues or released into the medium after subculture. This was confirmed through many indirect procedures (effect of exogenously added tyrosinase, induction of polyphenol oxidase [PPO] activity by auxin, etc.). The study of tyramine structure-activity relationships further suggested that the toxicity of tyramine might be due to the formation of indolequinones after oxidation by PPO. Subculture of calli grown on 2,4-dichlorophenoxyacetic acid in a medium containing benzyladenine triggered a slow decrease in PPO activity and dramatic increases in peroxidase and tyramine hydroxycinnamoyl transferase (THT) activities. THT was undetectable in calli grown on 2,4-dichlorophenoxyacetic acid but very active in tyramine-resistant crown gall cultures. Moreover, when [3H]tyramine was fed in vivo to tyramine-resistant tissues, it was rapidly integrated into cell walls in the wound periderm formed at the periphery of the calli. Both the conjugation of tyramine and its integration into cell walls could compete with the formation of toxic quinones and therefore play a part in the resistance. Thus, it seems likely that the control of the toxicity of hydroxyphenylethylamines by cytokinins results primarily from changes in the metabolism and the

  12. 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.

  13. Conifer tissue culture and how it may impact the pulp and paper industry

    SciTech Connect

    Verma, D.C.; Einspahr, D.W.

    1983-11-01

    This is a report on the state-of-the-art of tissue culture of conifers. Developments in organogenesis and somatic embryogenesis are looked at. This technology is expected to help the pulp and paper industry in achieving its goal of maximum productivity in two principal ways: (a) by providing rapid and efficient in vitro propagation methods for elite trees, and (b) by providing a technology for producing desired hybrids via somatic cell genetics and hybridization.

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

    PubMed

    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

    2015-11-13

    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.

  15. Tissue culture methods for the clonal propagation and genetic improvement of Spanish red cedar (Cedrela odorata).

    PubMed

    Peña-Ramírez, Yuri; Juárez-Gómez, Juan; González-Rodríguez, José Antonio; Robert, Manuel L

    2012-01-01

    The choice of a method to culture red cedar tissues depends on the final objectives pursued. If homogeneous clonal material is required for experimental purposes, the easiest way is to generate the lines through adventitious shoot induction from seedlings germinated from seeds. If the objective is to generate high yielding material for plantation purposes, the choice will be the same method but starting from mature vegetative tissues from selected elite plants. Most of the process are the same, but the initial steps are less efficient and much more elaborate. If the purpose is to generate lines with new genetic characteristics through somaclonal variation, mutagenesis, or genetic transformation, somatic embryogenesis will be required. No single method in its present form is suitable for all purposes. Eventually, the efficient production of somatic embryos from rejuvenated shoots collected from mature selected plants is the ideal way to culture this species, but for the time being we have to choose one or the other. In this chapter, we present a grafting procedure to rejuvenate and maintain mother plants in the greenhouse and the in vitro culture systems we have developed for the production of Cedrela odorata propagules using explants from both young seedlings and mature tissues from selected old trees. Using a modified TY17 medium and the BioMINT(®) temporary immersion system, we obtained high multiplication and ex vitro transplantation rates for efficient large-scale propagation of this species.

  16. 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.

  17. 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.

  18. An evolutionary view of plant tissue culture: somaclonal variation and selection.

    PubMed

    Wang, Qin-Mei; Wang, Li

    2012-09-01

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

  19. 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

  20. Comparison of four culture media for isolation of Mycobacterium avium complex from porcine tissues.

    PubMed

    Thoen, C O; Himes, E M; Jarnagin, J L; Harrington, R

    1979-02-01

    The efficiency of four culture media was compared for the isolation of Mycobacterium avium complex from 197 procine tissues. In 82 tissues with microscopic granulomas and acid-fast bacilli, a significantly greater number of isolates were obtained on Middlebrook 7H10 medium with sodium pyruvate than on Stonebrink medium, Herrold egg yolk agar medium, or Lowenstein-Jensen medium (P=0.01). In 46 tissues in which no microscopic granulomas or acid-fast bacilli were observed, a significantly greater number of isolates were made on Middlebrook 7H10 medium or Herrold egg yolk agar medium than on Stonebrink medium or on Lowenstein-Jensen medium (P=0.01). The time required to grow M. avium complex on Lowenstein-Jensen medium was significantly greater than the time required to observe growth on Stonebrink, Middlebrook 7H10, or Herrold egg yolk agar medium (p=0.001).

  1. Evaluation of osteoporosis prevention by adlay using a tissue culture model.

    PubMed

    Yang, Rong Sen; Chiang, Wenchang; Lu, Yi Hsiang; Liu, Shing Hwa

    2008-01-01

    Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf ) is a grass crop, which has been used in traditional Chinese medicine and also as a nourishing food. Recently, some studies have indicated that adlay possesses some pharmacological effects including anti-allergic, anti-mutagenic, hypolipemic, and anti-diabetic effects. However, the effect of adlay on osteoporosis is still unknown. In this study, we investigated and evaluated the effect of adlay seed on the osteoporosis prevention. The methods of in vitro cultures of neonatal rat calvaria tissues or adult rat femoral metaphyseal tissues of bones isolated from normal or ovariectomized female rats were used for further investigation. Treatment with water extract of adlay seed could reverse the decreased alkaline phosphatase activities and calcium levels and increased tartrate-resistant acidic phosphatase activities induced by parathyroid hormone in cultured metaphyseal tissues. In ovariectomized rats, the alkaline phosphatase activities and calcium levels were significantly decreased and tartrate-resistant acidic phosphatase activities were increased in femoral metaphyseal tissues as compared with sham-control. Treatment with water extract of adlay seed could counteract these effects in ovariectomized rats. Taken together, these findings imply that adlay is capable of reversing the osteoporotic status in rats, and may be a helpful healthy food for osteoporosis prevention.

  2. 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

  3. Association of tissue factor activity with the surface of cultured cells.

    PubMed Central

    Maynard, J R; Heckman, C A; Pitlick, F A; Nemerson, Y

    1975-01-01

    Tissue factor occurs in a dormant state on the surface of cultured normal human fibroblasts and WISH 1 amnion cells. The activity of undisturbed monolayers or cells lifted with brief trypsin treatment (0.125 per cent trypsin for 1 min) increases up to 60-fold upon prolonged digestion with dilute trypsin (0.0025 per cent trypsin for 30 min); activity appears subsequent to cell detachment. Up to 70 per cent of the total cellular tissue factor becomes active under these conditions and is released from the cells. The ruthenium red staining coat of the cells is lost during detachment, but cell viability (more than 90 per cent exclude trypan blue) and cell morphology do not change during the subsequent development of tissue factor activity. Furthermore, less than 10 percent of four intracellular enzymes and less than 20 per cent of two plasma membrane enzymes are released during this period of time. We therefore conclude that cells in culture do have tissue factor activity, that it exists in a latent form, and that total cell disruption is not necessary for this activity to initiate blood coagulation. Images PMID:47334

  4. 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.

  5. Cell Wall Regeneration around Protoplasts Isolated from Convolvulus Tissue Culture 1

    PubMed Central

    Horine, Randall K.; Ruesink, Albert W.

    1972-01-01

    Protoplasts of Convolvulus arvensis L. tissue culture regenerated a wall-like structure within 3 days in culture. Although unusually electron dense and atypically amorphous in the electron microscope, this structure could be digested with Myrothecium cellulase but was resistant to protease, a Rohm and Haas pectinase, and a β-1, 3-exoglucanase just like the original wall. A cytochemical test for callose was negative. Wall regeneration required a readily metabolized external carbon source and was not inhibited by a high concentration of cycloheximide, puromycin, or actinomycin D. Protoplast budding was correlated with the wall regeneration, and the latter was related quantitatively to the sucrose concentration in the medium. Although a concentration of 1 μm 2,4-dichlorophenoxy acetic acid is used normally for both general culture of the tissue and for wall regeneration, concentrations of 0 and 0.1 mm, which are highly deleterious to growth, have no appreciable effect on the incidence of the wall-like structure regenerated around protoplasts. The ability of protoplasts to undergo cell wall regeneration was decreased when they were cultured in the presence of proteolytic enzymes. Images PMID:16658192

  6. 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.

  7. Oxidation of External NAD(P)H by Mitochondria from Taproots and Tissue Cultures of Sugar Beet (Beta vulgaris).

    PubMed Central

    Zottini, M.; Mandolino, G.; Zannoni, D.

    1993-01-01

    The present study compares the exogenous NAD(P)H oxidation and the membrane potential ([delta][psi]) generated in mitochondria isolated from different tissues of an important agricultural crop, sugar beet (Beta vulgaris}. We observed that mitochondria from taproots, cold-stored taproots, and in vitro-grown tissue cultures contain a functional NADH dehydrogenase, whereas only those isolated from tissue cultures displayed a functional NAD(P)H dehydrogenase. It is interesting that the NADH-dependent [delta][psi] of mitochondria from cold-stored taproots and from tissue cultures was not affected by free Ca2+ ions, whereas free Ca2+ was required for the mitochondrial NADPH oxidation by in vitro-grown cells and cytosolic NADH oxidation by mitochondria from fresh taproots. A tentative model accounting for the different response to Ca2+ ions of the NADH dehydrogenase in mitochondria from cold-stored taproots and tissue cultures of B. vulgaris is discussed. PMID:12231847

  8. Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea

    PubMed Central

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

    2016-01-01

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

  9. Tissue Culture as a Source of Replicates in Nonmodel Plants: Variation in Cold Response in Arabidopsis lyrata ssp. petraea.

    PubMed

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

    2016-12-07

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

  10. Well Plate-Based Perfusion Culture Device for Tissue and Tumor Microenvironment Replication

    PubMed Central

    Zhang, W.; Gu, Y.; Hao, Y.; Sun, Q.; Konior, K.; Wang, H.

    2015-01-01

    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) 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

  11. Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

    PubMed

    Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2014-10-01

    The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

  12. 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.

  13. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    SciTech Connect

    Jewell, D.E.; Hausman, G.J.

    1986-03-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm/sup 2/ flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by (/sup 3/H)-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture.

  14. Native and recombinant bovine growth hormone antagonize insulin action in cultured bovine adipose tissue.

    PubMed

    Etherton, T D; Evock, C M; Kensinger, R S

    1987-08-01

    The current study was undertaken to determine if pituitary bovine GH (pbGH) and recombinant bGH (rbGH) antagonized insulin action in bovine adipose tissue after acute (2-h) and chronic (48-h) exposure and whether this was an intrinsic property of bGH. Insulin action (measured as the effect on incorporation of acetate-carbon into long-chain fatty acids) was unaffected by bGH in short term incubations regardless of whether hydrocortisone (HC) was present. After 48 h of culture, however, both pbGH and rbGH similarly antagonized the ability of insulin to maintain lipogenic capacity. This antagonism was dependent upon the presence of HC and was dose dependent, with half-maximal inhibition of insulin action occurring at about 0.5 ng/ml bGH. Bovine PRL did not mimic the effects of bGH on insulin action. These results establish that bGH antagonizes insulin action in bovine adipose tissue and that this effect is dependent upon long term exposure and the inclusion of HC in the culture medium. The fact that both rbGH and pbGH acted similarly indicates that this is an intrinsic property of bGH. The effect of bGH on insulin-dependent maintenance of lipogenic capacity may play an important role in redirecting nutrients away from adipose tissue to other tissues, such as muscle or mammary tissue. It is speculated that this metabolic effect of bGH plays an important role in the adaptive response to chronic bGH treatment, which increases milk yield of dairy cows and growth performance of beef cattle.

  15. Targeted metabolomics in cultured cells and tissues by mass spectrometry: method development and validation.

    PubMed

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

    2014-10-03

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

  16. Long term culture of epithelia in a continuous fluid gradient for biomaterial testing and tissue engineering.

    PubMed

    Minuth, W W; Strehl, R; Schumacher, K; de Vries, U

    2001-01-01

    Epithelia perform barrier functions being exposed to different fluids on the luminal and basal side. For long-term testing of new biomaterials as artificial basement membrane substitutes, it is important to simulate this fluid gradient. Individually-selected biomaterials can be placed in tissue carriers and in gradient containers, where different media are superfused. Epithelia growing on the tissue carriers form a physiological barrier during the whole culture period. Frequently however, pressure differences between the luminal and basal compartments occur. This is caused by a unilateral accumulation of gas bubbles in the container compartments resulting in tissue damage. Consequently, the occurence of gas bubbles has to be minimized. Air bubbles in the perfusion culture medium preferentially accumulate at sites where different materials come into contact. The first development is new screw caps for media bottles, specifically designed to allow fluid contact with only the tube and not the cap material. The second development is the separation of remaining gas bubbles from the liquid phase in the medium using newly-developed gas expander modules. By the application of these new tools, the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material can be significantly increased compared to earlier experiments.

  17. Enhancing plant regeneration in tissue culture: a molecular approach through manipulation of cytokinin sensitivity.

    PubMed

    Hill, Kristine; Schaller, G Eric

    2013-10-01

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

  18. Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.).

    PubMed

    Akello, Juliet; Dubois, Thomas; Gold, Clifford S; Coyne, Daniel; Nakavuma, Jessica; Paparu, Pamela

    2007-09-01

    Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.

  19. 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

  20. An electrochemical approach to monitor pH change in agar media during plant tissue culture.

    PubMed

    Wang, Min; Ha, Yang

    2007-05-15

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

  1. Tissue engineering of autologous cartilage grafts in three-dimensional in vitro macroaggregate culture system.

    PubMed

    Naumann, Andreas; Dennis, James E; Aigner, Joachim; Coticchia, James; Arnold, James; Berghaus, Alexander; Kastenbauer, Ernst R; Caplan, Arnold I

    2004-01-01

    In the field of tissue engineering, techniques have been described to generate cartilage tissue with isolated chondrocytes and bioresorbable or nonbioresorbable biomaterials serving as three-dimensional cell carriers. In spite of successful cartilage engineering, problems of uneven degradation of biomaterial, and unforeseeable cell-biomaterial interactions remain. This study represents a novel technique to engineer cartilage by an in vitro macroaggregate culture system without the use of biomaterials. Human nasoseptal or auricular chondrocytes were enzymatically isolated and amplified in conventional monolayer culture before the cells were seeded into a cell culture insert with a track-etched membrane and cultured in vitro for 3 weeks. The new cartilage formed within the in vitro macroaggregates was analyzed by histology (toluidine blue, von Kossa-safranin O staining), and immunohistochemistry (collagen types I, II, V, VI, and X and elastin). The total glycosaminoglycan (GAG) content of native and engineered auricular as well as nasal cartilage was assayed colorimetrically in a safranin O assay. The biomechanical properties of engineered cartilage were determined by biphasic indentation assay. After 3 weeks of in vitro culture, nasoseptal and auricular chondrocytes synthesized new cartilage with the typical appearance of hyaline nasal cartilage and elastic auricular cartilage. Immunohistochemical staining of cartilage samples showed a characteristic pattern of staining for collagen antibodies that varied in location and intensity. In all samples, intense staining for cartilage-specific collagen types I, II, and X was observed. By the use of von Kossa-safranin O staining a few positive patches-a possible sign of beginning mineralization within the engineered cartilages-were detected. The unique pattern for nasoseptal cartilage is intense staining for type V collagen, whereas auricular cartilage is only weakly positive for collagen types V and VI. Engineered nasal

  2. 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

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

    PubMed

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

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

  4. Increased oxidative stress in the placenta tissue and cell culture of tumour-bearing pregnant rats.

    PubMed

    Toledo, M T; Ventrucci, G; Gomes-Marcondes, M C C

    2011-11-01

    Placental dysfunction leads to foetal damage, which jeopardises the exchange between the maternal and foetal systems. We evaluated the effects of tumour growth on the activity of antioxidant enzymes and oxidative stress in placental tissue and cell culture from tumour-bearing pregnant rats compared to non-tumour-bearing pregnant rats that were ascitic fluid injected. Ascitic fluid is obtained from Walker tumour-bearing rats and contains a cytokine called Walker factor (WF), which is a molecule similar to proteolysis-inducing factor (PIF), and induces changes in protein metabolism and oxidative stress. Pregnant Wistar rats were distributed into control (C), tumour-bearing (W) and ascitic fluid injected (A) groups and were sacrificed on days 16, 19 and 21 of pregnancy to analyse the profile of enzyme activities (glutathione-S-transferase (GST), catalase (CAT), alkaline phosphatase (AP)) and malondialdehyde (MDA) content in placental tissue. Meanwhile, placenta samples from all groups were obtained on day 21, placed in primary culture and treated with WF for 72 h. The presence of tumour or ascitic fluid reduced the protein content of the placental tissue. On day 16 there was a significant reduction in AP activity in W rats, and on day 19, CAT activity and MDA content significantly increased. These results indicate that the presence of cancer decreased antioxidant enzyme capacity in the placenta, increasing the amount of oxidation in these cells, which may contribute to irreversible placental damage and compromisefoetal development. WF treatment induces similar changes in placental cells in primary culture, resulting in less cell viability and increased oxidative stress. These results indicate that WF, provided by the tumour or inoculation of ascitic fluid, has negative effects on placental homeostasis, which impairs foetal health.

  5. 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

  6. 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.

  7. 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.

  8. Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

    PubMed Central

    Lampl, Thomas; Crum, Jo A.; Davis, Taylor A.; Milligan, Carol; Del Gaizo Moore, Victoria

    2015-01-01

    Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput. PMID:25866954

  9. Monitoring fibroblast behavior in tissue culture with an applied electric field.

    PubMed Central

    Giaever, I; Keese, C R

    1984-01-01

    Mammalian fibroblasts have been cultured on evaporated gold electrodes subjected to an alternating electric field at 4000 Hz. The system consists of a large (approximately equal to 2 cm2) and a small (approximately equal to 3 X 10(-4) cm2) electrode bathed in tissue culture medium. The applied electric field produces a voltage drop at the boundary between the solution and the small electrode of a few mV at a current density of a few mA/cm2. The small population of cells that attach and spread on this electrode have a marked effect on the measured impedance and also cause it to fluctuate with time. The amplitude of these fluctuations is greatly reduced by cytochalasin B (10 microM), suggesting they are a consequence of cell movement. Images PMID:6587391

  10. 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.

  11. 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.

  12. Target detect system in 3D using vision apply on plant reproduction by tissue culture

    NASA Astrophysics Data System (ADS)

    Vazquez Rueda, Martin G.; Hahn, Federico

    2001-03-01

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

  13. 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.

  14. Cultured Human Adipose Tissue Pericytes and Mesenchymal Stromal Cells Display a Very Similar Gene Expression Profile

    PubMed Central

    Malta, Tathiane Maistro; de Deus Wagatsuma, Virgínia Mara; Palma, Patrícia Viana Bonini; Araújo, Amélia Goes; Ribeiro Malmegrim, Kelen Cristina; Morato de Oliveira, Fábio; Panepucci, Rodrigo Alexandre; Silva, Wilson Araújo; Kashima Haddad, Simone; Covas, Dimas Tadeu

    2015-01-01

    Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3+ lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols. PMID:26192741

  15. Cell culture models of higher complexity in tissue engineering and regenerative medicine.

    PubMed

    James Kirkpatrick, Charles; Fuchs, Sabine; Iris Hermanns, Maria; Peters, Kirsten; Unger, Ronald E

    2007-12-01

    Cell culture techniques have tended to be used in biomaterial research as a screening method prior to embarking on specific in vivo experimentation. This presentation aims at showing that it is possible to develop more sophisticated in vitro systems using primary human cells in co-culture with other cell types and biomaterials in a three-dimensional setting. While the predictive value of such systems is still not proven these models can be employed to unravel the complexity of biological systems in order to understand molecular mechanisms of cell-cell and cell-material interactions. The brief overview is under the headings of basic principles of relevant culture systems, the study of inflammation and the healing response, scenarios for specific biomaterial applications and future directions. How human endothelial cells can be usefully incorporated into more complex cell culture models is presented as an example of how relevant questions in tissue engineering and regenerative medicine can be addressed. The central tenet of this paper is that it is possible to refine in vitro methodology using cells of human origin to establish relevant assay systems that more closely simulate the cellular and molecular microenvironment encountered in a specific situation of regeneration using biomaterials.

  16. 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.

  17. Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

    PubMed

    Taylor, Drew W; Ahmed, Nazish; Hayes, Anthony J; Ferguson, Peter; Gross, Allan E; Caterson, Bruce; Kandel, Rita A

    2012-08-01

    To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

  18. 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

  19. Characterization of the Immunochemical Forms of Calcitonin Released by a Medullary Thyroid Carcinoma in Tissue Culture

    PubMed Central

    Goltzman, David; Tischler, Arthur S.

    1978-01-01

    Immunoreactive calcitonin released by a medullary thyroid carcinoma in tissue culture has been found to exhibit heterogeneity when analyzed by gel chromatography and radioimmunoassay, in a pattern analogous to that seen in the circulation of the patient from whom the neoplasm was removed. To examine the cause of the heterogeneity, the immunoreactive material released by the tumor into tissue culture medium was further analyzed by gel electrophoresis in the presence of the protein denaturant 8 M urea, by gel chromatography after reduction and alkylation, by affinity chromatography on concanavalin A-agarose, and by bioassay in a renal adenylyl cyclase system of enhanced sensitivity. The results suggest that the larger immunochemical forms of calcitonin described in the circulation of patients with medullary thyroid carcinoma may be released directly from the neoplasm and need not derive from peripheral metabolism of the monomer. It could be demonstrated that a major proportion of the immunochemical enlargement is dependent upon intermolecular disulfide bridge formation whereas aggregation or non-convalent protein binding account for a smaller component of the heterogeneity. In view of the absence of binding of the immunoreactive material to the lectin agarose, carbohydrate side chains, at least of the α-d glucosyl variety, do not seem to contribute significantly to calcitonin enlargement. Additionally, the studies indicate that, at least by in vitro assay, the larger immunochemical forms of calcitonin, representing the majority of the immunoreactivity released by a medullary thyroid carcinoma, are biologically inactive. PMID:621283

  20. Application of plant cell and tissue culture for the production of phytochemicals in medicinal plants.

    PubMed

    Pant, Bijaya

    2014-01-01

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

  1. Screenhouse and field persistence of nonpathogenic endophytic Fusarium oxysporum in Musa tissue culture plants.

    PubMed

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

    2008-04-01

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

  2. Studies on the response of ewes to live chlamydiae adapted to chicken embryos or tissue culture.

    PubMed Central

    Becerra, V M; Ata, F A; Storz, J

    1976-01-01

    Ewes infected before gestation with chicken embryo or tissue culture adapted chlamydial strain B-577 were challenge inoculated with the homologous strain at four to 18 weeks of gestation. The ewes responsed with group specific complement fixing antibody titers of 1:8 to 1:256 by the second week after initial infection. A secondary antibody response in the surviving challenge inoculated ewes occurred at the time of lambing and reached titers of 1:32 to 1:256 by the second week after parturition. Group specific complement fixing antibodies did not appear to play a significant role in resistance to chlamydial infection. Ewes infected with the chicken embryo adapted strain B-577 excreted chlamydiae in their feces 60 days after inoculation. However, chlamydiae were not recovered from feces of ewes infected with the tissue culture adapted strain B-577. Placentas of ewes challenge inoculated by the intravenous route were consistently infected. Chlamydiae were recovered from placentas, some fetuses and lambs. In two instances when challenge inoculation was given by the intramuscular route, infection was detected only by the direct fluorescent antibody method. PMID:1000377

  3. Finite element study of scaffold architecture design and culture conditions for tissue engineering.

    PubMed

    Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

    2009-10-01

    Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

  4. Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection.

    PubMed

    Zernova, Olga; Zhong, Wei; Zhang, Xing-Hai; Widholm, Jack

    2008-11-01

    This study was carried out to determine if the tobacco anthranilate synthase ASA2 2.3 kb promoter drives tissue culture specific expression and if it is strong enough to drive hpt (hygromycin phosphotransferase) gene expression at a level sufficient to allow selection of transformed soybean embryogenic culture lines. A number of transformed cell lines were selected showing that the promoter was strong enough. Northern blot analysis of plant tissues did not detect hpt mRNA in the untransformed control or in the ASA2-hpt plants except in developing seeds while hpt mRNA was detected in all tissues of the CaMV35S-hpt positive control line plants. However, when the more sensitive RT-PCR assay was used all tissues of the ASA2-hpt plants except roots and mature seeds were found to contain detectable hpt mRNA. Embryogenic tissue cultures initiated from the ASA2-hpt plants contained hpt mRNA detectable by both northern and RT-PCR analysis and the cultures were hygromycin resistant. Friable callus initiated from leaves of ASA2-hpt plants did in some cases contain hpt mRNA that was only barely detectable by northern hybridization even though the callus was very hygromycin resistant. Thus the ASA2 promoter is strong enough to drive sufficient hpt expression in soybean embryogenic cultures for hygromycin selection and only very low levels of expression were found in most plant tissues with none in mature seeds.

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

    PubMed

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

    1995-12-01

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

  6. Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells.

    PubMed

    Brovko, L; Minikh, O; Piekna, A; Griffiths, M W

    2011-07-01

    The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.

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

    PubMed Central

    Xie, Bing-Ying; Wu, Ai-Wen

    2016-01-01

    Background: Colorectal cancer (CRC) is a heterogeneous disease; current research relies on cancer cell lines and animal cancer models, which may not precisely imitate inner human tumors and guide clinical medicine. The purpose of our study was to explore and further improve the process of producing three-dimensional (3D) organoid model and impel the development of personalized therapy. Methods: We subcutaneously injected surgically resected CRC tissues from a patient into BALB/c-nu mice to build patient-derived xenografts (PDXs). Isolated cells from PDXs at appropriate tumor size were mingled with Matrigel, and then seeded in ultra-low attachment 96-well plates at four cell densities (500, 1000, 2000, and 4000 single cells/well). Cells were cultured with advanced Dulbecco's Modified Eagle Medium/F12 medium additional with various factors added to maintain tumor's biological traits and growth activity. The growth curves of the four cell densities were measured after 24 h of culture until 25 days. We evaluated the effects of four chemotherapeutic agents on organoid model by the CellTiter-Glo® Luminescent Cell Viability Assay. Hematoxylin and eosin (H and E) staining of 3D organoids was performed and compared with patient and CRC PDX tissues. Furthermore, immunohistochemistry was performed, in which the organoids were stained with the proliferation marker, Ki-67. During the experimental process, a phase-contrast microscope was used. Results: Phenotype experimental results showed that 3D organoids were tightly packed together and grew robustly over time. All four densities of cells formed organoids while that composed of 2000 cells/well provided an adequate cultivation system and grew approximately 8-fold at the 25th day. The chemosensitivity of the four conventional drugs was [s]-10-hydroxycamptothecin > mitomycin C > adriamycin > paclitaxel, which can guide clinical treatment. Histological features of CRC patient's tumor tissues and mice tumor xenograft tissues were

  8. Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

    PubMed

    Duressa, Tewodros Firdissa; Huybrechts, Roger

    2016-01-01

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

  9. Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon).

    PubMed Central

    Fuerst, J A; Sambhi, S K; Paynter, J L; Hawkins, J A; Atherton, J G

    1991-01-01

    During attempts to establish tissue cultures from hepatopancreas, heart, and hemolymph of the giant tiger prawn (Penaeus monodon), using a medium including penicillin, streptomycin, and amphotericin B, bacterial contamination in the form of a sheet of growth attached to the tissue culture vessel was a persistent problem. Contaminant bacteria were teardrop-shaped cells arranged in rosettes, and electron microscopy revealed buds, crateriform structures, and the absence of a peptidoglycan layer in the cell wall, features characteristic of bacteria in the Planctomyces-Pirellula group, a phylogenetically distinct group of eubacteria. Two strains of contaminant bacteria were isolated in pure culture. Both exhibited morphology and antibiotic resistance consistent with their membership in the Planctomyces-Pirellula group (order Planctomycetales) of eubacteria. Tissue culture media for marine invertebrates may select for such bacteria if high concentrations of cell wall synthesis-inhibiting antibiotics are included. Images PMID:1781677

  10. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    PubMed Central

    Shil, Sharadindu; Joshi, R. S.; Joshi, C. G.; Patel, A. K.; Shah, Ravi K.; Patel, Namrata; Jakhesara, Subhash J.; Kundu, Sumana; Reddy, Bhaskar; Koringa, P. G.; Rank, D. N.

    2017-01-01

    Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma) frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells. PMID:28246447

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

    PubMed Central

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

    2017-01-01

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

  12. Replication of type 2 herpes simplex virus in human endocervical tissue in organ culture.

    PubMed

    Birch, J; Fink, C G; Skinner, G R; Thomas, G H; Jordan, J A

    1976-08-01

    The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 37 degrees and in serum-supplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a "non-human" herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ.

  13. Replication of type 2 herpes simplex virus in human endocervical tissue in organ culture.

    PubMed Central

    Birch, J.; Fink, C. G.; Skinner, G. R.; Thomas, G. H.; Jordan, J. A.

    1976-01-01

    The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 37 degrees and in serum-supplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a "non-human" herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ. Images Fig. 1 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:183806

  14. Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

    PubMed

    Simain-Sato, F; Lahmouzi, J; Heinen, E; Defresne, M P; De Pauw-Gillet, M C; Grisar, T; Legros, J J; Legrand, R

    1999-08-01

    Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

  15. Quality control of cultured tissues requires tools for quantitative analyses of heterogeneous features developed in manufacturing process.

    PubMed

    Kino-Oka, Masahiro; Takezawa, Yasunori; Taya, Masahito

    2009-02-01

    Tissue engineering and related technology have attracted a great deal of medical attention as promising fields for curing defective tissues in vivo. Nowadays, many companies have been established for supplying the reconstructed grafts of cultured tissues for transplantation. The manufacturing processes generally deals with the handlings of starter cells offered by patients (or donors) as raw materials to cultured tissues as products, requiring the construction of novel ex vivo methodologies based on principles different from conventional processes for chemical and pharmaceutical productions. In addition, the raw materials have heterogeneity depending on the state of patients and location of cell harvests, and the products possess spatial cell distribution in the three dimensional structure. These features request a unique strategy in manufacturing process accompanied with the quality control for raw materials and products. This review article describes the contribution of tissue bankers and biochemical engineers to the quality control of cultured tissues during manufacturing, introducing the advances in methodologies to evaluate spatial heterogeneity of cells (or aggregates) and matrices in cultured tissues.

  16. 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.

  17. Amino acid pharmacology of mammalian central neurones grown in tissue culture.

    PubMed

    Barker, J L; Ransom, B R

    1978-07-01

    1. Spinal and cerebellar-brainstem areas of fetal mouse were dissociated and grown in tissue culture until large enough to permit stable intracellular recording. 2. The tissue-cultured neurones, growing as a monolayer and accessible under direct vision using phase contrast optics, allowed precise placement of intracellular recording and extracellular ionophoretic pipettes. 3. Ionophoresis of GABA and glutamate revealed a non-uniform distribution of responses over the cell surface, with a lack of spatial coincidence in sensitivity between the two. GABA inhibited and glutamate excited all cells tested. 4. GABA responses evoked at the cell body and on nearby process membrane were almost uniformly hyperpolarizing, while those at some peripheral process membrane were either hyperpolarizing, depolarizing or a combination of both events. All responses were associated with an increase in membrane slope conductance. 5. Membrane polarization showed that all hyperpolarizing events extrapolated to about the same inversion potential, which averaged about 9 mV more negative than resting potential (n = 95 cells). The depolarizing phases of responses evoked at peripheral membranes extrapolated to about 0 mV (n = 5 cells). 6. The hyperpolarization and increase in membrane conductance of GABA responses at the cell body were dependent on Cl- ions and the inversion potential of the response was dependent on the Cl- ion concentration gradient. The inversion potentials of GABA, glycine and beta-alanine responses were identical. 7. When matched in magnitude for evoked conductance increase, glycine responses decayed more rapidly than GABA. Glycine and beta-alanine voltage responses both decayed faster than GABA responses of comparable size. 8. In about half the cells tested sustained or rapidly repeated application of GABA and glycine transformed hyperpolarizing responses into depolarizations which were associated with a maintained conductance increase. Results from conditioning

  18. Measurement of Cu/Zn SOD in placenta, cultured cells, various fetal tissues, decidua and semen by ELISA.

    PubMed

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

    1998-07-01

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

  19. Experiments on tissue culture in the genus Lycopersicon miller : Shoot formation from protoplasts of tomato long-term cell cultures.

    PubMed

    Koblitz, H; Koblitz, D

    1982-06-01

    Callus cultures from cotyledon explants were established and maintained in culture for more than two years. After several months callus cultures were transferred into liquid medium and cultured as cell suspensions. Protoplasts were isolated from these cell suspension cultures and cultured in a liquid medium. After formation of new cell walls the cells were further cultured in liquid medium and afterwards transferred to an agar-solidified medium to give a vigorously growing callus culture. In the case of the cultivar 'Lukullus' shoots were recovered from callus. All efforts to root these shoots failed and this, in addition to variations in appearence, suggests that the shoots are changed genetically possibly due to the prolonged culture period.

  20. An in line non-invasive optical system to monitor pH in cell and tissue culture.

    PubMed

    Xu, Xia; Smith, Stanton; Urban, Jill; Cui, Zhanfeng

    2006-06-01

    pH is an important control parameter for maintenance of cell viability and for improving tissue functions during cell and tissue culture. pH monitoring during cell/tissue culture also provides valuable information on cell metabolic processes and living environment. In this study, an on-line, non-invasive pH monitoring system was developed for use during tissue/cell culture in a perfusion system, using an optical method. This device included light sources, light detectors, light guides and a flow cell. Phenol red was used as a pH indicator dye, and the ratio of light intensities at two wavelengths was measured at the same pH value. Low solution volume as low as around 10 microl could be used to detect pH. Compared to the conventional optical methods, this non-contact optical measurement can avoid the contamination of the tip of optical fibre during the long-term monitoring. It provides a possibility to do on-line monitoring and apply feed back control to maintain the culture environment at the desired conditions required for long-term cell/tissue culture.

  1. Short Term Culture of Vitrified Human Ovarian Cortical Tissue to Assess the Cryopreservation Outcome: Molecular and Morphological Analysis

    PubMed Central

    Ramezani, Mehdi; Salehnia, Mojdeh; Jafarabadi, Mina

    2017-01-01

    Background: The aim of the present study was to evaluate the effectiveness of human ovarian vitrification protocol followed with in vitro culture at the morphological and molecular levels. Methods: Ovarian tissues were obtained from 10 normal transsexual women and cut into small pieces and were divided into non-vitrified and vitrified groups and some of the tissues fragments in both groups were randomly cultured for two weeks. The morphological study using hematoxylin and eosin and Masson’s trichrome staining was done. The analysis of mean follicular density, 17-β estradiol (E2) and anti mullerian hormone (AMH), and real-time RT-PCR was down for the evaluation of expression of genes related to folliculogenesis. Data were compared by paired-samples and independent-samples T test. Values of p<0.05 were considered statistically significant. Results: The proportion of normal follicles did not show significant difference between vitrified and non-vitrified groups before and after culture but these rates and the mean follicle density significantly decreased in both cultured tissues (p<0.05). The expression of genes was similar in vitrified and non-vitrified groups but in cultured tissues the expression of GDF9 and FSHR genes increased and the expression of FIGLA and KIT-L genes decreased (p<0.05). An increase in E2 and AMH concentration was observed after 14 days of culture in both groups. Conclusion: In conclusion, the present study indicated that the follicular development and gene expression in vitrified ovarian tissue was not altered before and after in vitro culture, thus this method could be useful for fertility preservation; however, additional studies are needed to improve the culture condition. PMID:28377895

  2. A rapid method for fibronectin purification on nitrocellulose membranes suitable for tissue culture.

    PubMed

    Chifflet, Silvia; Bolatto, Carmen; Tolosa, Susana

    2004-05-31

    We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.

  3. Antibody against tuberlin: the specific visualization of cytoplasmic microtubules in tissue culture cells.

    PubMed Central

    Weber, K; Pollack, R; Bibring, T

    1975-01-01

    Cytoplasmic microtubules in tissue culture cells can be directly visualized by immunofluorescence microscopy. Antibody against tubulin from the outer doublets of sea urchin sperm flagella decorates a network of fine cytoplasmic fibers in a variety of cell lines of human, monkey, rat, mouse, and chicken origin. These fibers are separate and of uniform thickness and are seen throughout the cytoplasm. The fibers disappear either in a medium containing colchicine or after subjection of the cells to low temperature. The same treatments do not destroy the microfilamentous structures that are visualized by means of antibody against actin. When tryspin-treated enucleated cells are replated and then stained with antibody against tubulin, the fibers can be seen to traverse the entire enucleated cytoplasm. Images PMID:804694

  4. 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

  5. DNA polymorphism in Cab locus of tomato induced by tissue culture.

    PubMed

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

    1992-03-01

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

  6. 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.

  7. 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.

  8. 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.

  9. A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering.

    PubMed

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

    2010-11-01

    Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

  10. Detection of Border disease antigen in tissues of affected sheep and in cell cultures by immunofluorescence.

    PubMed

    Terpstra, C

    1978-11-01

    An account is presented of the distribution of fluorescence in cryostat sections of tissues from eight lambs with Border disease (BD). In young lambs fluorescence was observed in almost every organ, indicating a generalised infection with BD virus. Fluorescence was most prominent in the secretory glands of the alimentary and respiratory tracts, the basal cell layers of the epidermis and mucous membranes, and in the medullary rays of the kidneys. Abomasum, pancreas, kidneys, testicles and thyroid were most consistently affected. Although the number of fluorescing tissues decreased with age, viral antigen could still be detected in two sheep of 22 and 52 weeks old. Border disease viral antigen was demonstrated in cell cultures derived from the brain, kidney and testicle of six out of seven lambs despite the presence of neutralising antibody against bovine virus diarrhoea virus in three of them. The presence of the virus in the skin, the vascular walls and the endocrine system is discussed in relation to the aberrant development of fetal hair follicles, periarteritis and growth retardation respectively.

  11. 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

    2016-05-06

    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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Phenotype expression of gingival fibroblasts cultured on membranes used in guided tissue regeneration.

    PubMed

    Locci, P; Calvitti, M; Belcastro, S; Pugliese, M; Guerra, M; Marinucci, L; Staffolani, N; Becchetti, E

    1997-09-01

    Human gingival fibroblasts were cultured in vitro using as substrates an extracellular matrix (matrix) and polytetrafluoride (PTFE) membranes, which are used in guided tissue regeneration. To test the degree of biocompatibility of these membranes, the cellular proliferation and the accumulation of extracellular matrix (ECM) macromolecules were considered as parameters. The fibroblasts were cultured in vitro for 24 and 48 hours without serum on plastic, matrix, and PTFE membranes in the presence of 3H-thymidine, 3H-glucosamine, and 3H-proline to study the neo-synthesis of DNA, glycosaminoglycans (GAG), and collagen proteins, respectively. Studies on cell proliferation showed that fibroblasts grown on matrix membrane significantly increased 3H-thymidine incorporation, while fibroblasts grown on PTFE membrane decreased 3H-thymidine incorporation, compared to plastic used as a control. Moreover, the PTFE membrane induced a marked decrease of collagen and GAG accumulation both in the cellular and extracellular pool, while the matrix membrane provoked a decrease of the two macromolecules in the cellular pool and an increase in the extracellular one, compared to the control. The data we obtained demonstrate that matrix membranes are the most suitable to stimulate both cellular proliferation and ECM macromolecule accumulation.

  18. Plant tissue culture of fast-growing trees for phytoremediation research.

    PubMed

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

    2012-01-01

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

  19. Hydrogel network design using multifunctional macromers to coordinate tissue maturation in ovarian follicle culture

    PubMed Central

    Shikanov, Ariella; Smith, Rachel M; Xu, Min; Woodruff, Teresa K.; Shea, Lonnie D.

    2011-01-01

    Synthetic hydrogels with tunable properties are appealing for regenerative medicine. A critical limitation in hydrogel design at low solids concentration is the formation of defects, which increase gelation times and swelling, and reduce elasticity. Here, we report that tri-functional crosslinking peptides applied to 4-arm poly-(ethylene glycol) (PEG) hydrogels decreased swelling and gelation time relative to bi-functional crosslinkers. In contrast to bi-functional peptides, the third cross-linking site on the peptide created a branch point if an intramolecular crosslink formed, which prevented non-functional “dangling-ends” in the hydrogel network and enhanced the number of elastically active cross-links. The improved network formation enabled mouse ovarian follicle encapsulation and maturation in vitro. Hydrogels with bi-functional crosslinkers resulted in cellular dehydration, likely due to osmosis during the prolonged gelation. For tri-functional crosslinkers, the hydrogels supported a 17-fold volumetric expansion of the tissue during culture, with expansion dependent on the ability of the follicle to rearrange its microenvironment, which is controlled through the sensitivity of the cross-linking peptide to the proteolytic activity of plasmin. The improved network design enabled ovarian follicle culture in a completely synthetic system, and can advance fertility preservation technology for women facing premature infertility from anticancer therapies. PMID:21247629

  20. Gravity spun polycaprolactone fibres for soft tissue engineering: interaction with fibroblasts and myoblasts in cell culture.

    PubMed

    Williamson, Matthew Richard; Adams, Eric F; Coombes, Allan G A

    2006-03-01

    Poly(epsilon-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering.

  1. 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.

  2. Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells.

    PubMed Central

    Kooistra, T; van den Berg, J; Töns, A; Platenburg, G; Rijken, D C; van den Berg, E

    1987-01-01

    Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells. Images Fig. 2. Fig. 3. Fig. 5. Fig. 7. PMID:2827633

  3. Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues.

    PubMed

    Fortier, Guillaume Marceau; Gauvin, Robert; Proulx, Maryse; Vallée, Maud; Fradette, Julie

    2013-04-01

    Mesenchymal cells are central to connective tissue homeostasis and are widely used for tissue-engineering applications. Dermal fibroblasts and adipose-derived stromal cells (ASCs) allow successful tissue reconstruction by the self-assembly approach of tissue engineering. This method leads to the production of multilayered tissues, devoid of exogenous biomaterials, that can be used as stromal compartments for skin or vesical reconstruction. These tissues are formed by combining cell sheets, generated through cell stimulation with ascorbic acid, which favours the cell-derived production/organization of matrix components. Since media motion can impact on cell behaviour, we investigated the effect of dynamic culture on mesenchymal cells during tissue reconstruction, using the self-assembly method. Tissues produced using ASCs in the presence of a wave-like movement were nearly twice thicker than under standard conditions, while no difference was observed for tissues produced from dermal fibroblasts. The increased matrix deposition was not correlated with an increased proliferation of ASCs, or by higher transcript levels of fibronectin or collagens I and III. A 30% increase of type V collagen mRNA was observed. Interestingly, tissues engineered from dermal fibroblasts featured a four-fold higher level of MMP-1 transcripts under dynamic conditions. Mechanical properties were similar for tissues reconstructed using dynamic or static conditions. Finally, cell sheets produced using ASCs under dynamic conditions could readily be manipulated, resulting in a 2 week reduction of the production time (from 5 to 3 weeks). Our results describe a distinctive property of ASCs' response to media motion, indicating that their culture under dynamic conditions leads to optimized tissue engineering.

  4. Phenolic content in differentiated tissue cultures of untransformed and Agrobacterium-transformed roots of anise (Pimpinella anisum L.).

    PubMed

    Andarwulan, N; Shetty, K

    1999-04-01

    To investigate the role of differentiation of anise tissue cultures on total phenolic and anethole contents, benzylaminopurine- and thidiazuron-induced shoot cultures were generated from roots of the A-8 clonal line and its Agrobacterium rhizogenes-induced genetically transformed derivative JB-10. Embryogenic cultures were induced following 2,4-D treatment. Root cultures were multiplied on hormone-free medium. The effect of proline on differentiation and phenolic synthesis was also investigated. GC/MS studies indicate that anethole was not produced in root or other differentiated cultures. The predominant phenolic metabolite, however, was an anethole precursor, epoxypseudoisoeugenol-2-methylbutyrate (EPB). Total phenolics and EPB contents were highest in root cultures, which also correlated with higher proline content. Embryo and shoot cultures had reduced phenolic level and EPB and proline contents. Antioxidant activity in all differentiating cultures was high on day 60 compared to that on day 30, and there was no significant difference between differentiating tissues. This indicated that antioxidant protection might be linked not only to phenolics but to other nonphenolic metabolites as well.

  5. 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.

  6. 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.

  7. Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

    PubMed Central

    Shahin, Kifah; Doran, Pauline M.

    2011-01-01

    Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min−1) and gradually increasing (0.075–0.2 mL min−1) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0–4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8–5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min−1. GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention. PMID:21858004

  8. Characterization of the Embryogenic Tissue of the Norway Spruce Including a Transition Layer between the Tissue and the Culture Medium by Magnetic Resonance Imaging

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  9. 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.

  10. Inhibitory Effect of Progesterone on Cervical Tissue Formation in a Three-Dimensional Culture System with Human Cervical Fibroblasts1

    PubMed Central

    House, Michael; Tadesse-Telila, Serkalem; Norwitz, Errol R.; Socrate, Simona; Kaplan, David L.

    2013-01-01

    ABSTRACT Progesterone supplementation is recommended to prevent preterm birth in women with a short cervix, but the mechanism is unclear. We hypothesize that progesterone acts by altering the composition of the cervical extracellular matrix (ECM). We tested this hypothesis using human cervical fibroblasts in both two-dimensional (2D) and three-dimensional (3D) cultures. For 2D culture, cells were seeded in 6-well plates and cultured with media supplemented with estradiol (10−8 M), progesterone (10−7 or 10−6 M), and vehicle. For 3D culture, the cells were cultured on a porous silk protein scaffold system. Progesterone and estrogen receptors were documented by immunohistochemistry and Western blot analysis. In both 2D and 3D cultures, decreased collagen synthesis was seen with increased progesterone concentration. Three-dimensional cultures could be maintained significantly longer than 2D cultures, and the morphology of 3D cultures appeared similar to native cervical tissue. Thus, further studies were performed in 3D culture. To determine the effect of progesterone concentration, the 3D scaffolds were cultured with estradiol (10−8 M) and five conditions: vehicle; 10−9, 10−8, or 10−7 M progesterone; or 10−7 M progesterone plus 10−6 M mifepristone. The highest progesterone concentration correlated with the least amount of collagen synthesis. Collagen synthesis progressively increased as progesterone concentration decreased. This effect was partially antagonized by mifepristone, suggesting the mechanism is mediated by the progesterone receptor. This hormonally responsive 3D culture system supports the hypothesis that progesterone has a direct effect on remodeling cervical ECM during pregnancy. The 3D culture system could be useful for studying the mechanism of progesterone effects on the cervix. PMID:24285720

  11. Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers.

    PubMed

    May, S G; Savell, J W; Lunt, D K; Wilson, J J; Laurenz, J C; Smith, S B

    1994-12-01

    The primary objective of this study was to provide evidence for preadipocyte proliferation during culture of adipose tissue explants; a secondary objective was to compare the lipogenic activity and cellularity of adipose tissues from American Wagyu crossbred steers. Subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues were obtained at slaughter from the 2nd to 6th lumbar region of the loin from Angus (n = 10) and Wagyu crossbred steers (n = 10) that had been fed for 552 d by typical Japanese production standards. Adipose tissue explants were incubated 36 h with [3H]thymidine in the absence and presence of aphidicolin (a specific inhibitor of genomic DNA replication). Adipocytes were liberated by collagenase treatment and [3H]thymidine incorporation into DNA was measured. Whereas there were no significant differences between adipose tissue depots, Wagyu s.c. and i.m. preadipocytes and stromal-vascular cells exhibited greater (P < .05) [3H]thymidine incorporation into DNA than adipocytes from Angus steers. Intramuscular adipose tissue from both breeds exhibited lower (P < .05) rates of lipogenesis from acetate both before and after long-term (36-h) incubation than s.c. adipose tissue. Furthermore, i.m. adipocytes were smaller (P < .05) than s.c. adipocytes. The activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase were greater (P < .05) in Wagyu s.c. adipose tissue and less in Wagyu i.m. adipose tissue than in corresponding Angus tissues. There were no differences between breed types (P = .17) in rates of lipogenesis from acetate, either before or after explant culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Cytoenzymology and 3H-thymidine uptake of retro-ocular connective tissue cultures in experimental endocrino-exophthalmos.

    PubMed

    Vaida, E; Petrescu, R; Ghinea, E; Stefaneanu, L

    1976-01-01

    The in vitro retro-ocular connective tissue cultures from guinea pigs with endocrine exophthalmos were studied before and after retro-ocular treatment with cortisol and hyaluronidase. Both cortisol and hyaluronidase inhibited the cell proliferation, the cytoenzymic activities of oxydoreductases, the 3H-thymidine uptake, the number of mitoses and the protein content of cultivated cells.

  13. Quantitative analysis of toxin extracts from various tissues of wild and cultured puffer fish by an electrophysiological method.

    PubMed

    Sasaki, Keita; Takayama, Yasunori; Tahara, Tatsuo; Anraku, Kensaku; Ito, Yushi; Akaike, Norio

    2008-03-15

    We observed the effects of the toxin extracted from various tissues of wild and cultured puffer fish on voltage-dependent sodium current (I(Na)) using single rat CA1 neurons, and compared the results with that of tetrodotoxin (TTX). Toxin extracts from wild puffer fish inhibited I(Na) in a dilution-dependent manner, and toxin extracts from liver or ovary produced 300 times greater inhibition than that from muscle, and corresponded to about 65 microg TTX/g tissue. We also used puffer fish cultured in net cages or in tanks set up on land, in an attempt to isolate them from the food chain. The toxin extracts from cultured puffer fish also suppressed I(Na), but the inhibition was much weaker, and the effects of toxin extracts were almost the same in all tissues examined including liver, ovary, muscle, etc. We calculated the maximum edible amount for each tissue, assuming that the lethal dose of TTX is 1-10 microg/kg, and we found that the liver or ovary was edible in the case of cultured puffer fish.

  14. Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics.

    PubMed

    Vitrenko, Yakov; Kostenko, Iryna; Kulebyakina, Kateryna; Duda, Alla; Klunnyk, Mariya; Sorochynska, Khrystyna

    2017-02-16

    Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

  15. Root system architecture in Arabidopsis grown in culture is regulated by sucrose uptake in the aerial tissues.

    PubMed

    Macgregor, Dana R; Deak, Karen I; Ingram, Paul A; Malamy, Jocelyn E

    2008-10-01

    This article presents a detailed model for the regulation of lateral root formation in Arabidopsis thaliana seedlings grown in culture. We demonstrate that direct contact between the aerial tissues and sucrose in the growth media is necessary and sufficient to promote emergence of lateral root primordia from the parent root. Mild osmotic stress is perceived by the root, which then sends an abscisic acid-dependent signal that causes a decrease in the permeability of aerial tissues; this reduces uptake of sucrose from the culture media, which leads to a repression of lateral root formation. Osmotic repression of lateral root formation in culture can be overcome by mutations that cause the cuticle of a plant's aerial tissues to become more permeable. Indeed, we report here that the previously described lateral root development2 mutant overcomes osmotic repression of lateral root formation because of a point mutation in Long Chain Acyl-CoA Synthetase2, a gene essential for cutin biosynthesis. Together, our findings (1) impact the interpretation of experiments that use Arabidopsis grown in culture to study root system architecture; (2) identify sucrose as an unexpected regulator of lateral root formation; (3) demonstrate mechanisms by which roots communicate information to aerial tissues and receive information in turn; and (4) provide insights into the regulatory pathways that allow plants to be developmentally plastic while preserving the essential balance between aboveground and belowground organs.

  16. 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...

  17. 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

  18. 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.

  19. A total extract dot blot hybridization procedure for mRNA quantitation in small samples of tissues or cultured cells.

    PubMed

    Grimes, A; McArdle, H J; Mercer, J F

    1988-08-01

    A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit 10(5) cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression of the result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes.

  20. Qualitative and quantitative analysis of anthraquinone derivatives in rhizomes of tissue culture-raised Rheum emodi Wall. plants.

    PubMed

    Malik, Sonia; Sharma, Nandini; Sharma, Upendra K; Singh, Narendra P; Bhushan, Shashi; Sharma, Madhu; Sinha, Arun K; Ahuja, Paramvir S

    2010-06-15

    This paper presents quantification of five anthraquinone derivatives (emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion) in rhizomes of hardened micro-propagated Rheum emodi plants using high-performance liquid chromatography (HPLC). Aseptic shoot cultures were raised using rhizome buds. Shoot multiplication occurred in both agar gelled and liquid Murashige and Skoog (MS) medium supplemented with 10.0 microM 6-benzylaminopurine (BAP) and 5.0 microM indole-3-butyric acid (IBA). Rooted plantlets obtained on plant growth regulator (PGR)-free medium were transferred to soil with 92% survival. HPLC analysis revealed the presence of five anthraquinone derivatives: emodin glycoside, chrysophanol glycoside, emodin, chrysophanol and physcion in rhizomes of tissue culture-raised plants. Only emodin glycoside (1) and chrysophanol glycoside (2) were present in 6-month-old hardened tissue cultured plants. In addition, the other three derivatives (emodin (3), chrysophanol (4) and physcion (5)) were also detected after 9 months.

  1. Cell therapy, 3D culture systems and tissue engineering for cardiac regeneration.

    PubMed

    Emmert, Maximilian Y; Hitchcock, Robert W; Hoerstrup, Simon P

    2014-04-01

    Ischemic Heart Disease (IHD) still represents the "Number One Killer" worldwide accounting for the death of numerous patients. However the capacity for self-regeneration of the adult heart is very limited and the loss of cardiomyocytes in the infarcted heart leads to continuous adverse cardiac-remodeling which often leads to heart-failure (HF). The concept of regenerative medicine comprising cell-based therapies, bio-engineering technologies and hybrid solutions has been proposed as a promising next-generation approach to address IHD and HF. Numerous strategies are under investigation evaluating the potential of regenerative medicine on the failing myocardium including classical cell-therapy concepts, three-dimensional culture techniques and tissue-engineering approaches. While most of these regenerative strategies have shown great potential in experimental studies, the translation into a clinical setting has either been limited or too rapid leaving many key questions unanswered. This review summarizes the current state-of-the-art, important challenges and future research directions as to regenerative approaches addressing IHD and resulting HF.

  2. Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

    PubMed

    Kitajima, Takashi; Obuse, Sei; Adachi, Takahiro; Tomita, Masahiro; Ito, Yoshihiro

    2011-10-01

    The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.

  3. Cardioprotection by resveratrol: a review of effects/targets in cultured cells and animal tissues

    PubMed Central

    Wu, Joseph M; Hsieh, Tze-chen; Wang, Zhirong

    2011-01-01

    The most widespread form of cardiovascular disease (CVD) in the western world today is atherosclerosis (AS), notably of the major arteries. Despite decades of laboratory, animal, and clinical investigation providing multiple leads and intervention strategies that have resulted in a progressive decline in CVD-related mortality rates, the etiology of AS is incomplete; hence, the need for management and prevention remains imperative. AS is initiated by endothelial cell (EC) injury whereas its progression is promoted by EC interaction with cells that are recruited to the injured endothelium, and additionally, bioactive cytokines and chemokines elaborated by the intercellular interplay. A primary research interest of our laboratory has been the mechanisms that underlie the “French paradox” - an epidemiological association of the co-existence of risk factors with low CVD incidence/mortality rates postulated to attribute to low-to-moderate consumption of red wine and grape-derived polyphenol resveratrol. This review summarizes effects and targets of resveratrol in cultured human aortic and pulmonary aortic endothelial cells (HAEC and HPAEC) and animal tissues, with focus on the resveratrol target protein RTP, N-ribosyldihydro-nicotinamide:quinone oxidoreductase (NQO2). PMID:22254184

  4. Plant cell, tissue and organ culture: the most flexible foundations for plant metabolic engineering applications.

    PubMed

    Ogita, Shinjiro

    2015-05-01

    Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially regarding the production of bioactive plant secondary metabolites and metabolic engineering in target plants are discussed mainly from the phenotypic view point.

  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.

  6. 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.

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

    PubMed

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

    2013-01-01

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

  8. Human dental pulp stem cells cultured onto dentin derived scaffold can regenerate dentin-like tissue in vivo.

    PubMed

    Tran, Ha Le Bao; Doan, Vu Nguyen

    2015-12-01

    Regeneration of dentin tissues in the pulp space of teeth serves the ultimate goal of preserving teeth via endodontic approaches. In recent times, many studies suggested that human dentin scaffolds combined with dental stem cells was a potential strategy for the complete dentin tissue regeneration. In this study, human dental pulp stem cells (DPSCs) were isolated and cultured. Dentin specimens were prepared from human third molars and treated with ethylene diamine tetra-acetic acid and citric acid to remove the smear layer. Then, DPSCs were cultured onto human treated dentin (hTD) and implanted in mouse model for 4, 6 and 8 weeks. The resulting grafts were assessed by hematoxylin and eosin stain and immunohistochemical stains. As a result, DPSCs were supported and induced to regenerate of dentin-like tissues which expressed specific dentin markers such as dentin sialophosphoprotein and dentin matrix protein 1 by combination with hTD in vivo. Furthermore, cells existed in the newly-formed dentin-like tissues also expressed typical human mitochondria antibodies, demonstrated that new tissues originated from human. In conclusion, the obtain results extend hopefully newly-established therapy to apply in endodontics and traumatic dental hard tissues.

  9. Tissue Harvesting Site and Culture Medium Affect Attachment, Growth, and Phenotype of Ex Vivo Expanded Oral Mucosal Epithelial Cells.

    PubMed

    Islam, Rakibul; Eidet, Jon Roger; Badian, Reza A; Lippestad, Marit; Messelt, Edward; Griffith, May; Dartt, Darlene A; Utheim, Tor Paaske

    2017-04-06

    Transplantation of cultured oral mucosal epithelial cells (OMECs) is a promising treatment strategy for limbal stem cell deficiency. In order to improve the culture method, we investigated the effects of four culture media and tissue harvesting sites on explant attachment, growth, and phenotype of OMECs cultured from Sprague-Dawley rats. Neither choice of media or harvesting site impacted the ability of the explants to attach to the culture well. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM) and Roswell Park Memorial Institute 1640 medium (RPMI) supported the largest cellular outgrowth. Fold outgrowth was superior from LL explants compared to explants from the buccal mucosa (BM), HP, and transition zone of the lower lip (TZ) after six-day culture. Putative stem cell markers were detected in cultures grown in DMEM and RPMI. In DMEM, cells from TZ showed higher colony-forming efficiency than LL, BM, and HP. In contrast to RPMI, DMEM both expressed the putative stem cell marker Bmi-1 and yielded cell colonies. Our data suggest that OMECs from LL and TZ cultured in DMEM give rise to undifferentiated cells with high growth capacity, and hence are the most promising for treatment of limbal stem cell deficiency.

  10. Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

    PubMed

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

    2015-11-01

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

  11. Effects of frutalin on early follicle morphology, ultrastructure and gene expression in cultured goat ovarian cortical tissue.

    PubMed

    Soares, Maria A A; Costa, José J N; Vasconcelos, Gisvani L; Ribeiro, Regislane P; Souza, José C; Silva, André L C; Van den Hurk, Robert; Silva, José R V

    2017-02-15

    Frutalin is a galactose-binding lectin that has an irreversible cytotoxic effect on HeLa cervical cancer cells, by inducing apoptosis and inhibiting cell proliferation. It was previously shown that after in vitro incubation, frutalin is internalized into HeLa cells nucleus, which indicates that frutalin apoptosis-inducing activity might be linked with its nuclear localization. Considering that drugs commonly used for cancer treatment have a deleterious effect on germ cells, the aim of this study was to evaluate the effect of frutalin on the activation, survival, ultrastructure and gene expression in follicles cultured within ovarian tissue. Goat ovarian fragments were cultured for 6 days in α-MEM+ alone or supplemented with frutalin (1, 10, 50, 100 or 200 µg/ml). Non-culturad and cultured tissues were processed for histological and ultrastructural analysis and they were also stored to evaluate the expression of anti- and pro-apoptotic genes by quantitative polymerase chain reaction (qPCR). The results showed that the frutalin, at all concentrations tested, reduced follicular survival when compared with control medium. Higher concentrations of frutalin (50, 100 or 200 µg/ml) also reduced follicular survival when compared with those tissues cultured with 1 or 10 µg/ml of frutalin. The ultrastructural analysis showed that atretic cultured follicles had retracted oocytes and a large number of vacuoles spread throughout the cytoplasm. In addition, signs of damage of mitochondrial membranes and cristae were observed. Moreover, although a dose-response effect on gene expression has not been observed, when compared with tissues culture in control medium, the presence of frutalin increased in mRNA expression pro-apoptotic genes. In conclusion, frutalin reduces follicular survival at all concentrations tested, its effects being more pronounced when high concentrations of this lectin (50, 100 and 200 µg/ml) are used. Gene expression profile and ultrastrutural features of

  12. Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.).

    PubMed

    Lees, G L

    1986-08-01

    Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.

  13. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

    PubMed

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid; Grivel, Jean-Charles

    2009-12-01

    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

  14. Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications.

    PubMed

    Neelakandan, Anjanasree K; Wang, Kan

    2012-04-01

    In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.

  15. PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Uzal, F A; Hugenholtz, P; Blackall, L L; Petray, S; Moss, S; Assis, R A; Fernandez Miyakawa, M; Carloni, G

    2003-02-02

    The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

  16. Flow cytometric analysis of the cell cycle in different coconut palm (Cocos nucifera L.) tissues cultured in vitro.

    PubMed

    Sandoval, A; Hocher, V; Verdeil, J-L

    2003-08-01

    We conducted a study of the cell cycle of coconut palm tissues cultured in vitro in order to regulate regeneration. Coconut palm is a plant for which it is difficult to monitor the ability of the meristematic cells to actively divide. Cell nuclei were isolated from various types of coconut palm tissues with and without in vitro culture. After the nuclei were stained with propidium iodide, relative fluorescence intensity was estimated by flow cytometry. Characterization of the cell cycle reinforced the hypothesis of a block in the G(0)/G(1) and G(1)/S phases of the coconut cells. A time-course study carried out on immature leaves revealed that this block takes place gradually, following the introduction of the material in vitro. Synchronization of in vitro-cultured leaves cells using 60 micro M aphidicholin revealed an increase in the number of nuclei in the S phase after 108 h of treatment. The significance of these results is discussed in relation with the ability of coconut tissue cultured in vitro to divide.

  17. Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve

    PubMed Central

    Huang, Lanfeng; Li, Rui; Liu, Wanguo; Dai, Jin; Du, Zhenwu; Wang, Xiaonan; Ma, Jianchao; Zhao, Jinsong

    2014-01-01

    Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, but cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhesion stage. Moreover, seeded cells were distributed throughout the material. PMID:25221594

  18. Intracellular Production of Brucella L Forms II. Induction and Survival of Brucella abortus L Forms in Tissue Culture1

    PubMed Central

    Hatten, Betty A.; Sulkin, S. Edward

    1966-01-01

    Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. II. Induction and survival of Brucella abortus L forms in tissue culture. J. Bacteriol. 91:14–20. 1966.—Intracellular survival of altered brucellae, possibly L forms, was not greatly affected by penicillin or streptomycin in concentrations ranging from 5.0 to 40 μg/ml, but a combination of these two antibiotics (2.5 to 20 μg/ml each) reduced the number of positive L-form cultures. Tetracycline (2.0 μg/ml) decreased the number of positive L-form cultures at about the same rate as combinations of the higher concentrations of penicillin and streptomycin. Various concentrations of tetracycline (0.1 to 2.0 μg/ml) with 5.0 μg/ml of penicillin or streptomycin significantly reduced the number of positive L-form cultures. L forms were recovered for several days after elimination of bacteria from the cultures by all of the antibiotics tested. L-form production was not dependent upon the presence of antibiotics in the culture medium, but they were recovered in greater numbers when bacteria were still present in the hamster kidney cells. Addition of thallium acetate to infected cells (at varying intervals of time after infection) to control bacterial growth and conversion to the L phase during cellular disintegration decreased the number of positive L-form cultures obtained over a 10-day period. Comparison of the antibiotic sensitivity of bacteria recovered from infected tissue culture cells with the stock strain of Brucella abortus indicated that some resistance to penicillin and tetracycline had developed. A marked resistance to streptomycin was observed in those bacteria recovered from cells maintained in the presence of this antibiotic. PMID:4955246

  19. Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

    PubMed

    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.

  20. Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells.

    PubMed Central

    Drake, T. A.; Hannani, K.; Fei, H. H.; Lavi, S.; Berliner, J. A.

    1991-01-01

    Oxidatively modified low-density lipoprotein is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis through its biologic effects on vascular cells. This study examined the effects of minimally oxidized preparations of LDL (MM-LDL) on tissue factor (TF) expression by cultured human endothelial cells. Low-density lipoprotein purified from normal donors was modified by exposure to iron or by prolonged storage, resulting in levels of thiobarbituric acid-reacting substances of approximately 2.5 to 4 nmoles/mg cholesterol. Preparations had less than 2.5 pg of endotoxin per microgram LDL and had no intrinsic procoagulant activity. This form of modified but not native LDL induced TF expression in endothelial cells in a time- and dose-dependent manner. Peak TF coagulant activity in cells exposed to 40 micrograms/ml MM-LDL were observed at 4 to 6 hours, and ranged from 50 to 500 pg/10(5) cells, compared with less than 10 pg/10(5) cells exposed to native LDL. Northern blot analysis showed TF mRNA levels to increase approximately 30-fold with exposure to MM-LDL for 2 hours. Induction of TF activity was dependent on the concentration of MM-LDL from 1 microgram/ml to 80 micrograms/ml, a range in which cell viability and morphology were unaffected. The findings suggest that minimally oxidized LDL may be a local mediator promoting thrombosis in atherosclerotic lesions. Images Figure 1 PMID:2000938

  1. Novel sterol glucosyltransferase in the animal tissue and cultured cells: evidence that glucosylceramide as glucose donor.

    PubMed

    Akiyama, Hisako; Sasaki, Narie; Hanazawa, Shuwa; Gotoh, Mari; Kobayashi, Susumu; Hirabayashi, Yoshio; Murakami-Murofushi, Kimiko

    2011-05-01

    Cholesteryl glucoside (CG), a membrane glycolipid, regulates heat shock response. CG is rapidly induced by heat shock before the activation of heat shock transcription factor 1 (HSF1) and production of heat shock protein 70 (HSP70), and the addition of CG in turn induces HSF1 activation and HSP70 production in human fibroblasts; thus, a reasonable correlation is that CG functions as a crucial lipid mediator in stress responses in the animal. In this study, we focused on a CG-synthesizing enzyme, animal sterol glucosyltransferase, which has not yet been identified. In this study, we describe a novel type of animal sterol glucosyltransferase in hog stomach and human fibroblasts (TIG-3) detected by a sensitive assay with a fluorescence-labeled substrate. The cationic requirement, inhibitor resistance, and substrate specificity of animal sterol glucosyltransferase were studied. Interestingly, animal sterol glucosyltransferase did not use uridine diphosphate glucose (UDP-glucose) as an immediate glucose donor, as has been shown in plants and fungi. Among the glycolipids tested in vitro, glucosylceramide (GlcCer) was the most effective substrate for CG formation in animal tissues and cultured cells. Using chemically synthesized [U-((13))C]Glc-β-Cer as a glucose donor, we confirmed by mass spectrometry that [U-((13))C]CG was synthesized in hog stomach homogenate. These results suggest that animal sterol glucosyltransferase transfers glucose moiety from GlcCer to cholesterol. Additionally, using GM-95, a mutant B16 melanoma cell line that does not express ceramide glucosyltransferase, we showed that GlcCer is an essential substrate for animal sterol glucosyltransferase in the cell.

  2. An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate

    PubMed Central

    Krupkova, Olga; Hlavna, Marian; Amir Tahmasseb, Julie; Zvick, Joel; Kunz, Dominik; Ito, Keita; Ferguson, Stephen J.; Wuertz-Kozak, Karin

    2016-01-01

    Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1–20 U/mL) or by the diffusion of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1β and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease. PMID:27689996

  3. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    PubMed Central

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

    2016-01-01

    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  4. Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro.

    PubMed

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

    2005-12-01

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

  5. 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.

  6. Generation of strip-format fibrin-based engineered heart tissue (EHT).

    PubMed

    Schaaf, Sebastian; Eder, Alexandra; Vollert, Ingra; Stöhr, Andrea; Hansen, Arne; Eschenhagen, Thomas

    2014-01-01

    This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately ¼ of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

  7. Embryonic Stem Cell-Derived Neurons are a Novel, Highly Sensitive Tissue Culture Platform for Botulinum Research

    DTIC Science & Technology

    2011-01-01

    drug discovery while dramatically decreasing animal use. Published by Elsevier Inc. 1. Introduction The Clostridium botulinum neurotoxins (BoNTs) are...Quinn, Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells, Neurotoxicology 22 (2001) 447–453...tissue culture platform for botulinum research 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) McNutt, P, Celver, J, Hamilton, T, Mesngon

  8. New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures.

    PubMed

    El Tahchy, Anna; Boisbrun, Michel; Ptak, Agata; Dupire, François; Chrétien, Françoise; Henry, Max; Chapleur, Yves; Laurain-Mattar, Dominique

    2010-01-01

    Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d(3)-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling.

  9. Royal jelly prevents osteoporosis in rats: beneficial effects in ovariectomy model and in bone tissue culture model.

    PubMed

    Hidaka, Saburo; Okamoto, Yoshizo; Uchiyama, Satoshi; Nakatsuma, Akira; Hashimoto, Ken; Ohnishi, S Tsuyoshi; Yamaguchi, Masayoshi

    2006-09-01

    Royal jelly (RJ) has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham), ovariectomized (OVX), OVX given 0.5% (w/w) raw RJ, OVX given 2.0% (w/w) RJ, OVX given 0.5% (w/w) protease-treated RJ (pRJ), OVX given 2.0% (w/w) pRJ, OVX given 17beta-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD) by 24%. Administration of 17beta-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w) RJ and 0.5-2.0% (w/w) pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17beta-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH)-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  10. Fast isolation and expansion of multipotent cells from adipose tissue based on chitosan-selected primary culture.

    PubMed

    Huang, Guo-Shiang; Tseng, Ting-Chen; Dai, Niann-Tzyy; Fu, Keng-Yen; Dai, Lien-Guo; Hsu, Shan-Hui

    2015-10-01

    Adipose-derived adult stem cells (ASCs) have gained much attention because of their multipotency and easy access. Here we describe a novel chitosan-based selection (CS) system instead of the conventional plastic adherence (PA) to obtain the primary ASCs. The minimal amount of adipose tissue for consistent isolation of ASCs is reduced from 10 mL to 5 mL. The selection is based on the specific interaction between cells and chitosan materials, which separate ASCs by forming spheroids during primary culture. The primary culture period was reduced from 4 days to one day and more ASCs (ten-fold expansion) were achieved in a week. The average duration for obtaining 1 × 10(7) cells takes about seven days from 5 mL of adipose tissue, compared to 14 days using the conventional PA method from 10 mL of adipose tissue. The replicative senescence of CS-ASCs is not evident until the fifteenth passage (vs. eighth for the PA-ASCs). The obtained ASCs (CS-ASCs) have less doubling time for the same passage of cells and show greater stemness than those obtained from the conventional PA method (PA-ASCs). Moreover, CS-ASCs undergo trilineage differentiation more effectively than PA-ASCs. The greater differentiation potential of CS-ASCs may be associated with the enrichment and maintenance of CD271 positive cells by chitosan selection of primary culture.

  11. Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue.

    PubMed

    Hammond, S Austin; Carew, Amanda C; Helbing, Caren C

    2013-01-01

    Nanoparticles (NPs), materials that have one dimension less than 100 nm, are used in manufacturing, health, and food products, and consumer products including cosmetics, clothing, and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs), thyroxine, and 3,3',5-triiodothyronine (T3), are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs). We used the cultured tailfin (C-fin) assay to examine the exposure effects of 0.1-10 nM (~8-800 ng/L) of three types of ~20 nm TiO2 NPs (P25, M212, M262) and micron-sized TiO2 (μ TiO2) ±10 nM T3. The actual Ti levels were 40.9-64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR) was used to measure the relative amounts of mRNA transcripts encoding TH-responsive THs receptors (thra and thrb) and Rana larval keratin type I (rlk1), as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30), superoxide dismutase (sod), and catalase (cat). The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25, and M212 while no effect was observed with M262 exposure. Therefore, the risk of adversely affecting amphibian tissue by disrupting TH-signaling or inducing cellular stress is low for these compounds relative to other previously-tested NPs.

  12. Evaluation of the effects of titanium dioxide nanoparticles on cultured Rana catesbeiana tailfin tissue

    PubMed Central

    Hammond, S. Austin; Carew, Amanda C.; Helbing, Caren C.

    2013-01-01

    Nanoparticles (NPs), materials that have one dimension less than 100 nm, are used in manufacturing, health, and food products, and consumer products including cosmetics, clothing, and household appliances. Their utility to industry is derived from their high surface-area-to-volume ratios and physico-chemical properties distinct from their bulk counterparts, but the near-certainty that NPs will be released into the environment raises the possibility that they could present health risks to humans and wildlife. The thyroid hormones (THs), thyroxine, and 3,3′,5-triiodothyronine (T3), are involved in development and metabolism in vertebrates including humans and frogs. Many of the processes of anuran metamorphosis are analogous to human post-embryonic development and disruption of TH action can have drastic effects. These shared features make the metamorphosis of anurans an excellent model for screening for endocrine disrupting chemicals (EDCs). We used the cultured tailfin (C-fin) assay to examine the exposure effects of 0.1–10 nM (~8–800 ng/L) of three types of ~20 nm TiO2 NPs (P25, M212, M262) and micron-sized TiO2 (μ TiO2) ±10 nM T3. The actual Ti levels were 40.9–64.7% of the nominal value. Real-time quantitative polymerase chain reaction (QPCR) was used to measure the relative amounts of mRNA transcripts encoding TH-responsive THs receptors (thra and thrb) and Rana larval keratin type I (rlk1), as well as the cellular stress-responsive heat shock protein 30 kDa (hsp30), superoxide dismutase (sod), and catalase (cat). The levels of the TH-responsive transcripts were largely unaffected by any form of TiO2. Some significant effects on stress-related transcripts were observed upon exposure to micron-sized TiO2, P25, and M212 while no effect was observed with M262 exposure. Therefore, the risk of adversely affecting amphibian tissue by disrupting TH-signaling or inducing cellular stress is low for these compounds relative to other previously-tested NPs. PMID

  13. Isolation and characterization of diosgenin from in vitro cultured tissues of Helicteres isora L.

    PubMed

    Deshpande, Harshal A; Bhalsing, Sanjivani R

    2014-01-01

    The objective of the present study was to isolate, characterize, quantify and compare the accumulation of bioactive secondary metabolite-diosgenin from in vitro cultured cells of Helicteres isora and plant parts. The levels of this secondary compound were examined by using various biochemical techniques. The result showed that maximum diosgenin was obtained from in vitro cultured cells as compared to the plant parts. The fallout of this study is important, since levels of diosgenin detected in the in vitro cultured cells were more than in the plant parts. In vitro cultured cells accumulate comparatively higher amount of diosgenin making Helicteres isora a potentially new and indigenous source of diosgenin.

  14. Development of a Bioreactor to Culture Tissue Engineered Ureters Based on the Application of Tubular OPTIMAIX 3D Scaffolds.

    PubMed

    Seifarth, Volker; Gossmann, Matthias; Janke, Heinz Peter; Grosse, Joachim O; Becker, Christoph; Heschel, Ingo; Artmann, Gerhard M; Temiz Artmann, Aysegül

    2015-01-01

    Regenerative medicine, tissue engineering and biomedical research give hope to many patients who need bio-implants. Tissue engineering applications have already been developed based on bioreactors. Physiological ureter implants, however, do not still function sufficiently, as they represent tubular hollow structures with very specific cellular structures and alignments consisting of several cell types. The aim of this study was to a develop a new bioreactor system based on seamless, collagenous, tubular OPTIMAIX 3D prototype sponge as scaffold material for ex-vivo culturing of a tissue engineered ureter replacement for future urological applications. Particular emphasis was given to a great extent to mimic the physiological environment similar to the in vivo situation of a ureter. NIH-3T3 fibroblasts, C2C12, Urotsa and primary genitourinary tract cells were applied as co-cultures on the scaffold and the penetration of cells into the collagenous material was followed. By the end of this study, the bioreactor was functioning, physiological parameter as temperature and pH and the newly developed BIOREACTOR system is applicable to tubular scaffold materials with different lengths and diameters. The automatized incubation system worked reliably. The tubular OPTIMAIX 3D sponge was a suitable scaffold material for tissue engineering purposes and co-cultivation procedures.

  15. Empirical models to identify mechanisms driving reductions in tissue mercury concentration during culture of farmed southern bluefin tuna Thunnus maccoyii.

    PubMed

    Balshaw, S; Edwards, J W; Ross, K E; Ellis, D; Padula, D J; Daughtry, B J

    2008-12-01

    Two empirical models are presented to elucidate the mechanisms driving reductions in the mercury concentration of southern bluefin tuna (SBT) during culture. Model 1 predicts temporal fluctuations in mercury concentration in response to growth dilution. Model 2 predicts the combined effects of growth dilution and linear mercury accumulation. Model 2 was found to be the more accurate model. Over a typical farming period of 136 days, growth dilution resulted in a reduction in mean mercury concentration of SBT edible tissues from 0.51 mg/kg down to 0.33 mg/kg. Extended culture beyond 136 days resulted in an increase in mercury concentration due to the combined effects of mercury accumulation and seasonal lipid depletion. Results indicate that under current industry practice, cultured SBT can be consumed twice as frequently as that of wild caught SBT while maintaining total dietary mercury intake below national recommendations.

  16. Adipose tissue can be generated in vitro by using adipocytes from human fat tissue mesenchymal stem cells seeded and cultured on fibrin gel sheet.

    PubMed

    Tran, Cong Toai; Huynh, Duy Thao; Gargiulo, Ciro; Tran, Le Bao Ha; Huynh, Minh Hang; Nguyen, Khanh Hoa; Filgueira, Luis; Strong, D Micheal

    2013-03-01

    The current study has developed an innovative procedure to generate ex novo fat tissue by culturing adipocytes from human fat tissue mesenchymal stem cells (hFTMSCs) on fibrin gel sheet towards applications in medicine and cosmetology. Fibrin gel has been obtained by combining two components fibrinogen and thrombin collected by human peripheral blood. By this procedure it was possible to generate blocks of fibrin gel containing adipocytes within the gel that show similar features and consistency to human fat tissue mass. Results were assessed by histological staining methods, fluorescent immune-histochemistry staining as well photos by scanning electron microscopy (SEM) to demonstrate the adhesion and growth of cells in the fibrin gel. This result opens a real possibility for future clinical applications in the treatment of reconstructive and regenerative medicine where the use of stem cell may eventually be a unique solution or in the field of aesthetic medicine where autograft fat stem cells may grant for a safer and better outcome with long lasting results.

  17. Effects of temperature generated from the Holmium: YAG laser on human osteoblasts in monolayer tissue culture.

    PubMed

    Hafez, Moustafa I; Sandison, Anne; Coombs, Richard R H; McCarthy, Ian D; Hafez, Al-Shymaa M

    2012-01-01

    With the use of lasers for ablation purposes in spinal surgery, the tissue temperature increases above the boiling point of water, leading to tissue ablation by vaporisation. Due to the thermal environment engendered by the use of lasers, there is concern about the safety of the surrounding important structures, such as dura mater, dorsal root ganglia, and nerve roots.

  18. Real architecture For 3D Tissue (RAFT™) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells.

    PubMed

    Szebeni, Gabor J; Tancos, Zsuzsanna; Feher, Liliana Z; Alfoldi, Robert; Kobolak, Julianna; Dinnyes, Andras; Puskas, Laszlo G

    2017-04-01

    There is an unmet medical need for the improvement of pancreatic islet maintenance in culture. Due to restricted donor availability it is essential to ameliorate islet viability and graft engraftment. The aim of this study was to compare the standard tissue culture techniques with the advanced Real Architecture For 3D Tissue (RAFT™) culture system in terms of viability and hormone production. Here, we first report that islets embedded in RAFT™ collagen type I advanced tissue culture system maintain their tissue integrity better than in monolayer and suspension cultures. The Calcein violet assay and Annexin V/propidium-iodide staining show higher cell viability in the RAFT™ culture system. Quantitative real-time PCR data showed that RAFT™ increases insulin expression after 18 days in culture compared to traditional methods. Enhanced insulin and glucagon production was further verified by immunofluorescent staining in a time-course manner. These results indicate that RAFT™ tissue culture platform can be a promising tool to maintain pancreatic islet spheroid integrity and culture islets for downstream high throughput pharmacological studies ex vivo.

  19. Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique.

    PubMed

    Yamamoto, Yasunori; Ito, Akira; Kato, Masahiro; Kawabe, Yoshinori; Shimizu, Kazunori; Fujita, Hideaki; Nagamori, Eiji; Kamihira, Masamichi

    2009-12-01

    Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering.

  20. 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.

  1. Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus1

    PubMed Central

    Hatten, Betty A.; Sulkin, S. Edward

    1966-01-01

    Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285–296. 1966.—Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO2 was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus. Images PMID:16562102

  2. 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.

  3. 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 ...

  4. Occurrence of an Inhibitor of Tissue-Type Plasminogen Activator in Seeds and in Vitro Cultures of Erythrina caffra Thunb.

    PubMed

    Meyer, H J; van Staden, J

    1991-08-01

    The level of an inhibitor of tissue-type plasminogen activator (t-PA) increased slowly during the early developmental stage of seeds of Erythrina caffra Thunb. Thereafter, the inhibitor increased exponentially until the seeds reached maturity. At maturity, the t-PA inhibitor levels in the cotyledons were 38 times higher than the levels at the onset of seed development. The t-PA inhibitor accumulated at a faster rate than the storage proteins, which reached a concentration 15 times higher than the protein concentration at the onset of seed development. During the imbibition and germination process, the t-PA inhibitor decreased gradually. The inhibitor kept on decreasing during the growth of the seedlings until the 10th day after imbibition, when it leveled off at 4.1% of that of the initial inhibitor concentration. The inhibitor remained at this level until the cotyledons were shed at day 22. The total protein in the cotyledons decreased at a slower rate than the inhibitor and reached a minimum concentration at day 20 of 3.6% of the initial protein concentration in the cotyledons. Callus cultures of root, shoot, leaf, and cotyledonary tissue was established and maintained on Murashige-Skoog medium supplemented with 3% sucrose, 10 micromolar benzyladenine, and 5 micromolar 2,4-dichlorophenoxyacetic acid. A shoot cell suspension culture was established on Murashige-Skoog medium supplemented with 3% sucrose, 1 micromolar benzyladenine, and 0.5 micromolar 2,4-dichlorophenoxyacetic acid (pH 5.7) and shaken at 60 revolutions per minute. The level of t-PA inhibitor in root, shoot, leaf, and cotyledonary callus was substantially lower than in the corresponding intact tissue. The t-PA inhibitor levels in the linear growth phase was higher than in the lag or stationary growth phases of the cell suspension culture. A hydrolysate of the cell walls of tomato and E. caffra Thunb, as well as polyamines and organic acids, did not increase the concentration of t-PA inhibitor in

  5. 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.

  6. 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

  7. Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

    DTIC Science & Technology

    2013-06-01

    compatible bioreactor optimized in year 1 to identify hyperpolarized metabolic biomarkers of prostate cancer presence and aggressiveness. To...accomplish this goal my group finished the engineering of a 5 mm bioreactor and acquired hyperpolarized [1-13C]pyruvate data indicating that similar signal...to noise and quality data can be achieved with 4 to 5 prostate tissue slices in the 5 mm bioreactor as was acquired from 30-40 tissue slices in the

  8. Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

    DTIC Science & Technology

    2013-06-01

    bioreactor optimized in year 1 to identify hyperpolarized metabolic biomarkers of prostate cancer presence and aggressiveness. To accomplish this goal my...group finished the engineering of a 5 mm bioreactor and acquired hyperpolarized [1-13C]pyruvate data indicating that similar signal to noise and...quality data can be achieved with 4 to 5 prostate tissue slices in the 5 mm bioreactor as was acquired from 30-40 tissue slices in the prior 10 mm

  9. Microfluidics and cancer analysis: cell separation, cell/tissue culture, cell mechanics, and integrated analysis systems.

    PubMed

    Pappas, Dimitri

    2016-01-21

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

  10. 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

  11. Quantitative techniques for assessing and controlling the dispersion and biological effects of multiwalled carbon nanotubes in mammalian tissue culture cells.

    PubMed

    Wang, Xiang; Xia, Tian; Ntim, Susana Addo; Ji, Zhaoxia; George, Saji; Meng, Huan; Zhang, Haiyuan; Castranova, Vincent; Mitra, Somenath; Nel, André E

    2010-12-28

    In vivo studies have demonstrated that the state of dispersion of carbon nanotubes (CNTs) plays an important role in generating adverse pulmonary effects. However, little has been done to develop reproducible and quantifiable dispersion techniques to conduct mechanistic studies in vitro. This study was to evaluate the dispersion of multiwalled carbon nanotubes (MWCNTs) in tissue culture media, with particular emphasis on understanding the forces that govern agglomeration and how to modify these forces. Quantitative techniques such as hydrophobicity index, suspension stability index, attachment efficiency, and dynamic light scattering were used to assess the effects of agglomeration and dispersion of as-prepared (AP), purified (PD), or carboxylated (COOH) MWCNTs on bronchial epithelial and fibroblast cell lines. We found that hydrophobicity is the major factor determining AP- and PD-MWCNT agglomeration in tissue culture media but that the ionic strength is the main factor determining COOH-MWCNT suspendability. Bovine serum albumin (BSA) was an effective dispersant for MWCNTs, providing steric and electrosteric hindrances that are capable of overcoming hydrophobic attachment and the electrostatic screening of double layer formation in ionic media. Thus, BSA was capable of stabilizing all tube versions. Dipalmitoylphosphatidylcholine (DPPC) provided additional stability for AP-MWCNTs in epithelial growth medium (BEGM). While the dispersion state did not affect cytotoxicity, improved dispersion of AP- and PD-MWCNTs increased TGF-β1 production in epithelial cells and fibroblast proliferation. In summary, we demonstrate how quantitative techniques can be used to assess the agglomeration state of MWCNTs when conducting mechanistic studies on the effects of dispersion on tissue culture cells.

  12. Isolation, in vitro culture and identification of a new type of mesenchymal stem cell derived from fetal bovine lung tissues.

    PubMed

    Hu, Pengfei; Pu, Yabin; Li, Xiayun; Zhu, Zhiqiang; Zhao, Yuhua; Guan, Weijun; Ma, Yuehui

    2015-09-01

    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.

  13. 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.

  14. Quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation.

    PubMed

    Zhang, Xinsheng; Hasoksuz, Mustafa; Spiro, David; Halpin, Rebecca; Wang, Shiliang; Vlasova, Anastasia; Janies, Daniel; Jones, Leandro R; Ghedin, Elodie; Saif, Linda J

    2007-06-20

    The genetic diversity of 2 pairs (AH65 and AH187) of wild type bovine coronaviruses (BCoV) sequenced directly from nasal (respiratory) and rectal (enteric) swabs of two feedlot calves with respiratory and enteric symptoms [Hasoksuz, M., Sreevatsan, S., Cho, K.O., Hoet, A.E., Saif, L.J., 2002b. Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res. 84 (1-2), 101-109.]. was analyzed. Sequence analysis of the complete genomes revealed differences at 123 and 149 nucleotides (nt) throughout the entire genome between the respiratory and enteric strains for samples AH65 and AH187, respectively, indicating the presence of intra-host BCoV quasispecies. In addition, significant numbers of sequence ambiguities were found in the genomes of some BCoV-R and BCoV-E strains, suggesting intra-isolate quasispecies. The tissue culture (TC) passaged counterparts of AH65 respiratory BCoV (AH65-R-TC) and enteric BCoV (AH65-E-TC) were also sequenced after 14 and 15 passages and 1 plaque purification in human rectal tumor cells (HRT-18), respectively. Compared to the parental wild type strains, tissue culture passage generated 104 nt changes in the AH65-E-TC isolate but only 8 nt changes in the AH65-R-TC isolate. Particularly noteworthy, the majority of nucleotide changes in the AH65-E-TC isolate occurred at the identical positions as the mutations occurring in the AH65-R strain from the same animal. These data suggest that BCoV evolves through quasispecies development, and that enteric BCoV isolates are more prone to genetic changes and may mutate to resemble respiratory BCoV strains after tissue culture passage.

  15. 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.

  16. 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.

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

    PubMed

    Campbell, Jonathan J; Davidenko, Natalia; Caffarel, Maria M; Cameron, Ruth E; Watson, Christine J

    2011-01-01

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

  18. Basic procedures for epigenetic analysis in plant cell and tissue culture.

    PubMed

    Rodríguez, José L; Pascual, Jesús; Viejo, Marcos; Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Yrei, Norma Yague; Santamaría, María E; Pérez, Marta; Fernández Fraga, Mario; Berdasco, María; Rodríguez Fernández, Roberto; Cañal, María J

    2012-01-01

    In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.

  19. Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches

    PubMed Central

    Singh, Pritika; Guleri, Rupam; Angurala, Amrita; Kaur, Kuldeep; Kaur, Kulwinder; Kaul, Sunil C.; Wadhwa, Renu

    2017-01-01

    Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field. PMID:28299323

  20. Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches.

    PubMed

    Singh, Pritika; Guleri, Rupam; Angurala, Amrita; Kaur, Kuldeep; Kaur, Kulwinder; Kaul, Sunil C; Wadhwa, Renu; Pati, Pratap Kumar

    2017-01-01

    Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

  1. 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.

  2. Coculture strategies in bone tissue engineering: the impact of culture conditions on pluripotent stem cell populations.

    PubMed

    Janardhanan, Sathyanarayana; Wang, Martha O; Fisher, John P

    2012-08-01

    The use of pluripotent stem cell populations for bone tissue regeneration provides many opportunities and challenges within the bone tissue engineering field. For example, coculture strategies have been utilized to mimic embryological development of bone tissue, and particularly the critical intercellular signaling pathways. While research in bone biology over the last 20 years has expanded our understanding of these intercellular signaling pathways, we still do not fully understand the impact of the system's physical characteristics (orientation, geometry, and morphology). This review of coculture literature delineates the various forms of coculture systems and their respective outcomes when applied to bone tissue engineering. To understand fully the key differences between the different coculture methods, we must appreciate the underlying paradigms of physiological interactions. Recent advances have enabled us to extrapolate these techniques to larger dimensions and higher geometric resolutions. Finally, the contributions of bioreactors, micropatterned biomaterials, and biomaterial interaction platforms are evaluated to give a sense of the sophistication established by a combination of these concepts with coculture systems.

  3. 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 ...

  4. 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...

  5. 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...

  6. 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.

  7. Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients

    PubMed Central

    Wu, L; Sluiter, A A; Guo, Ho-fu; Balesar, R A; Swaab, D F; Zhou, Jiang-Ning; Verwer, R W H

    2008-01-01

    Abstract Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control participants with rat embryonic day 14 (E14) neural stem cells. Viability staining based on the exclusion of ethidium bromide by intact plasma membranes showed that there were strikingly more viable cells and fewer dead cells in slices co-cultured with neural stem cells than in untreated slices. The presence of Alzheimer pathology in the brain slices did not influence this effect, although the slices from Alzheimer patients, in general, contained fewer viable cells. Co-culturing with rat E14 fibroblasts did not improve the viability of neurons in the human brain slices. Since the human slices and neural stem cells were separated by a membrane during co-culturing our data show for the first time that neural stem cells release diffusible factors that may improve the survival of aged and degenerating neurons in human brains. PMID:18088384

  8. 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...

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. Effects of dopamine and dopamine-active compounds on oxytocin and vasopressin production in rat neurohypophyseal tissue cultures.

    PubMed

    Gálfi, M; Janáky, T; Tóth, R; Prohászka, G; Juhász, A; Varga, C; László, F A

    2001-04-02

    The effects of dopamine (DA) or DA-active drugs on the synthesis of neurohypophyseal (NH) hormones were studied in 13-14 day cultures of isolated NH tissue from rats. The following DA-active compounds were used (10(-6) M in each medium): DA, apomorphine (APM), Pro-Lys-Gly (PLG), butaclamol (B), haloperidol (HP), chlorpromazine (CPZ) and sulpiride (SP). The oxytocin (OT) and vasopressin (VP) contents of the condensed media were determined by RIA after a 1 or 2 h incubation. Significantly increased contents of OT and VP were detected in the tissue culture media following DA, APM or PLG administration. This elevation of NH hormone production could be blocked by previous administration of B or the DA receptor antagonists HP, CPZ or SP. The application of B after DA agonists proved ineffective. The results indicate that NH hormone production can be directly influenced by the DA-ergic system. The DA-ergic control of NH hormone secretion in rats can occur independently of the hypothalamus, at the level of the posterior pituitary.

  14. Repair of Segmental Bone Defect Using Totally Vitalized Tissue Engineered Bone Graft by a Combined Perfusion Seeding and Culture System

    PubMed Central

    Feng, Ya-Fei; Li, Xiang; Hu, Yun-Yu; Wang, Zhen; Ma, Zhen-Sheng; Lei, Wei

    2014-01-01

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

  15. Immunodetection of Human LINE-1 Expression in Cultured Cells and Human Tissues.

    PubMed

    Sharma, Reema; Rodić, Nemanja; Burns, Kathleen H; Taylor, Martin S

    2016-01-01

    Long interspersed element-1 (LINE-1) is the only active protein-coding retrotransposon in humans. It is not expressed in somatic tissue but is aberrantly expressed in a wide variety of human cancers. ORF1p protein is the most robust indicator of LINE-1 expression; the protein accumulates in large quantities in cellular cytoplasm. Recently, monoclonal antibodies have allowed more complete characterizations of ORF1p expression and indicated potential for developing ORF1p as a clinical biomarker. Here, we describe a mouse monoclonal antibody specific for human LINE-1 ORF1p and its application in immunofluorescence and immunohistochemistry of both cells and human tissues. We also describe detection of tagged LINE-1 ORF2p via immunofluorescence. These general methods may be readily adapted to use with many other proteins and antibodies.

  16. Growth of Cowdria ruminantium in tissue culture endothelial cell lines from wild African mammals.

    PubMed

    Smith, G E; Anderson, E C; Burridge, M J; Peter, T F; Mahan, S M

    1998-04-01

    Endothelial cell cultures were established from several wild African mammalian species. Long-term cultures were established from three ruminants, stable antelope (Hippotragus niger), buffalo (Syncerus caffer), and eland (Tragelaphus oryx), and from an omnivore, the bushpig (Potamochoerus porcus). Cowdria ruminanntium was isolated from plasma of clinically affected animals in these four cell lines and in bovine endothelial cells used routinely for C. ruminantium propagation. Nineteen different strains of C. ruminantium from Africa and the Caribbean region were grown and maintained in these cell lines and their growth was comparable with growth in the bovine endothelial cells. The role of sable antelope, eland, and bushpigs in the epidemiology of heartwater is unknown. However, these results extend the number of cell lines that can be used to isolate and grow C. ruminantium.

  17. Ascorbic acid in cultured tissue of briar rose, Rosa rugosa Thunb.

    PubMed

    Wegg, S M; Townsley, P M

    1983-04-01

    Ascorbic acid synthesis was studied using suspension cultured cells from the fleshy portion of the fruit of the rose, Rosa rugosa Thunb. Cells cultured in the dark contained ascorbic acid at a level of 13 mg/100 g wet wt. This value increased several fold when the cells were grown under light. Ascorbic acid does not accumulate in the growth medium possibly due to its rapid oxidation. L-galactono-1,4-lactone was readily converted to ascorbic acid whereas L-gulono-1,4-lactone was used to a much lesser degree. D-glucose, D-fructose, D-galactose. D-glucurono-1,4-lactone and myoinositol did not stimulate increased ascorbic acid synthesis. Reducing the sucrose content of the medium reduced the ascorbic acid content of the cells without altering cell yield.

  18. Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

    DTIC Science & Technology

    2011-06-01

    Ronen’s report) above the rotor and pushing the tissues out the sample tube into the rotor using a small plastic rod. Following the insertion of each...for pathology and LDH enzyme activity. Two pathologists used a five-point scale (1= excellent, 5 = poor) to quantify the quality of the pathology of...AUC), time to maximum hyperpolarized lactate, maximum hyperpolarized lactate peak, and LDH activity were statistically compared between benign and

  19. Growth Stimulation by Catecholamines in Plant Tissue/Organ Cultures 1

    PubMed Central

    Protacio, Calixto M.; Dai, Yao-ren; Lewis, Eldrin F.; Flores, Hector E.

    1992-01-01

    Addition of catecholamines at micromolar concentrations caused a dramatic stimulation of growth of tobacco (Nicotiana tabacum) thin cell layers (TCLs) and Acmella oppositifolia “hairy” root cultures. A threefold increase in the rate of ethylene evolution was observed in the catecholamine-treated explants. Aminooxyacetic acid and silver thiosulfate, inhibitors of ethylene biosynthesis and action, respectively, reduced the growth-promoting effect of dopamine. However, these compounds alone could also inhibit the growth of the TCL explants. When ethylene in the culture vessel was depleted by trapping with mercuric perchlorate, dopamine-stimulated growth was still obtained, suggesting that ethylene does not mediate the dopamine effect. Dopamine potentiated the growth of TCLs grown in Murashige and Skoog medium supplemented with indoleacetic acid (IAA) and kinetin. When IAA was replaced by 2,4-dichlorophenoxyacetic acid, dopamine addition showed no growth-promoting effect. Instead, 2,4-dichlorophenoxyacetic acid stimulated the growth of TCL explants to the same extent as that obtained with IAA plus dopamine. Because synthetic auxins do not appear to be substrates for IAA oxidizing enzymes, we hypothesized that catecholamines exert their effect by preventing IAA oxidation. Consistent with this explanation, dopamine (25 micromolar) inhibited IAA oxidase activity by 60 to 100% in crude enzyme extracts from tobacco roots and etiolated corn coleoptiles, but had no effect on peroxidase activity in the same extracts. Furthermore, addition of dopamine to TCL cultures resulted in a fourfold reduction in the oxidative degradation of [1-14C]IAA fed to the explants. Because the growth enhancement by catecholamines is observed in both IAA-requiring and IAA-independent cultures, we suggest that these aromatic amines may have a role in the regulation of IAA levels in vivo. ImagesFigure 2 PMID:16668653

  20. Human Periodontal Cells Demonstrate Osteoblast-Like and Fibroblast-Like Characteristics in Tissue Culture

    DTIC Science & Technology

    1989-05-05

    this study was to characterize the phenotypes of cells cultured from human periodontium in order to provide insight into healing mechanisms ...migration using membranes is best suited to isolated defects 4 Figure 1. Models of Wound Healing Figure 1A. Cellular Mechanisms Mesenchymal stem cells... mechanisms and improve the clinical success of regenerative procedures. Listgarten (1986) further sug- gests wound healing is significant to the progression

  1. Using Xenopus tissue cultures for the study of myasthenia gravis pathogenesis.

    PubMed

    Yeo, Hwee Li; Lim, Jorain Yu Ni; Fukami, Yuki; Yuki, Nobuhiro; Lee, Chi Wai

    2015-12-15

    Myasthenia gravis (MG), the most common autoimmune disease of neuromuscular junction (NMJ), is heterogeneous in terms of pathophysiology, which is determined by the pathogenic antigen of autoantibodies targeting to synaptic proteins at the NMJs. Currently, patients suspected with MG are routinely screened for the presence of autoantibodies against acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) using a cell-based assay (CBA) that involves the expression of target synaptic membrane protein in heterologous cell lines. However, some autoantibodies may only show reactivity for binding to densely clustered AChR in the physiological conformation, while AChR clustering is known to involve signaling events orchestrated by over a dozen of postsynaptic proteins. To improve the existing serological diagnosis of MG, this study explored the possibility of using the well-established Xenopus primary culture system as a novel CBA for MG. Here, by examining the pathogenic effects of four MG human plasma samples, we found that the samples from both seropositive and seronegative MG patients effectively induced the disassembly of aneural AChR clusters in cultured Xenopus muscle cells, as well as the nerve-induced AChR clusters in the nerve-muscle co-cultures. Importantly, the disassembly of AChR clusters was spatio-temporally correlated to the disappearance of actin depolymerizing factor (ADF)/cofilin, an actin regulator involved in AChR trafficking and clustering. Taken together, this study develops a reliable CBA using Xenopus primary cultures for screening the pathogenicity of human MG plasma samples, and providing a platform for investigating the pathogenic mechanisms underlying the endocytic trafficking and degradation of AChRs at NMJs in MG patients.

  2. 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.

  3. High isoflavone content and estrogenic activity of 25 year-old Glycine max tissue cultures.

    PubMed

    Federici, Ermanno; Touché, André; Choquart, Sandie; Avanti, Ornella; Fay, Laurent; Offord, Elizabeth; Courtois, Didier

    2003-10-01

    Soy isoflavones are phytoestrogens which have been associated with several health benefits. In the present study, we report the production of isoflavones in a collection of 40 strains of soya cell cultures established in 1975. A large variability in the isoflavone composition was observed and high-producing strains, with an isoflavone content of up to 46.3 mg g(-1) dry wt., were found. In comparison with soybeans, many callus strains had a higher isoflavone concentration (10-40 times) and a different ratio of genistin to daidzin forms. The highest producing strain was transferred to liquid medium in an Erlenmeyer flask and in a 10 l stirred-tank bioreactor where high isoflavone content (7% dry wt.), concentration (880 mg l(-1)) and a maximum productivity estimated to 60 mg l(-1) d(-1) were obtained. We further studied the estrogenic activity of pure compounds compared to plant cell culture extracts in the estrogen-responsive human endometrial Ishikawa cell line. Estrogen was confirmed to be 1000-10,000 times more active than isoflavones. The estrogenic activity of the extracts correlated to their isoflavone content. The activity of the malonyl isoflavones, assessed here for the first time, was lower than the aglycones. Taken together, these results suggest that soya cell cultures can be used as an alternative source to soybeans to provide high concentrations of bioactive isoflavones.

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

    PubMed

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

    2014-01-01

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

  5. 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

  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. 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

  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. Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats.

    PubMed

    Goyal, Ritu; Guvendiren, Murat; Freeman, Onyi; Mao, Yong; Kohn, Joachim

    2017-01-11

    The design of composite tissue scaffolds containing an extracellular matrix (ECM) and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000), medium (E0500), and fast (E1000) degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw) retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days) was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days) did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days) allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds.

  10. Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

    PubMed Central

    Xu, Wei-Wei; Huang, Li; Chong, Kelvin K.L.; Leung, Doreen S.Y.; Li, Benjamin F.L.; Yin, Zheng-Qin; Huang, Yi-Fei; Pang, Chi Pui

    2017-01-01

    AIM To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells (ADSCs) generated by enzyme (EN) and explant (EX) culture methods. METHODS We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent (IMF) staining. RESULTS Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method. PMID:28149772

  11. Optimization of Polymer-ECM Composite Scaffolds for Tissue Engineering: Effect of Cells and Culture Conditions on Polymeric Nanofiber Mats

    PubMed Central

    Goyal, Ritu; Guvendiren, Murat; Freeman, Onyi; Mao, Yong; Kohn, Joachim

    2017-01-01

    The design of composite tissue scaffolds containing an extracellular matrix (ECM) and synthetic polymer fibers is a new approach to create bioactive scaffolds that can enhance cell function. Currently, studies investigating the effects of ECM-deposition and decellularization on polymer degradation are still lacking, as are data on optimizing the stability of the ECM-containing composite scaffolds during prolonged cell culture. In this study, we develop fibrous scaffolds using three polymer compositions, representing slow (E0000), medium (E0500), and fast (E1000) degrading materials, to investigate the stability, degradation, and mechanics of the scaffolds during ECM deposition and decellularization, and during the complete cellularization-decell-recell cycle. We report data on percent molecular weight (% Mw) retention of polymeric fiber mats, changes in scaffold stiffness, ECM deposition, and the presence of fibronectin after decellularization. We concluded that the fast degrading E1000 (Mw retention ≤ 50% after 28 days) was not sufficiently stable to allow scaffold handling after 28 days in culture, while the slow degradation of E0000 (Mw retention ≥ 80% in 28 days) did not allow deposited ECM to replace the polymer support. The scaffolds made from medium degrading E0500 (Mw retention about 60% at 28 days) allowed the gradual replacement of the polymer network with cell-derived ECM while maintaining the polymer network support. Thus, polymers with an intermediate rate of degradation, maintaining good scaffold handling properties after 28 days in culture, seem best suited for creating ECM-polymer composite scaffolds. PMID:28085047

  12. 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.

  13. [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.

  14. A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function.

    PubMed

    Wang, Xiuli; Sun, Lin; Maffini, Maricel V; Soto, Ana; Sonnenschein, Carlos; Kaplan, David L

    2010-05-01

    Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel/collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein alpha- and -beta mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.

  15. 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.

  16. The cytotoxic effects of asbestos and other mineral dust in tissue culture cell lines.

    PubMed Central

    Chamberlain, M.; Brown, R. C.

    1978-01-01

    The cytotoxic effects of 15 dusts have been tested in 2 cell lines. Experimental animal data are available for 11 of the 15 dusts. There is a correlation between the cytotoxic activities of the dusts and the ability to induce mesothelial tumours following intrapleural injection of the dusts. Some preliminary observations on the nature of interaction between the dusts, medium components and the cells are reported. It is suggested that the study of the interactions between cells and dusts in culture may lead to an understanding of the pathogenesis of these dusts in man and animals. Images Fig. 2 PMID:656318

  17. 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.

  18. Potential of tissue culture for breeding root-knot nematode resistance into vegetables.

    PubMed

    Fassuliotis, G; Bhatt, D P

    1982-01-01

    Plant protoplast technology is being investigated as a means of transferring root-knot nematode resistance factors from Solanum sisymbriifolium into the susceptible S. melongena. Solanum sisymbriifolium plants regenerated from callus lost resistance to Meloidogyne javanica but retained resistance to M. incognita. Tomato plants cloned from leaf discs of the root-knot nematode resistant 'Patriot' were completely susceptible to M. incognita, while sections of stems and leaves rooted in sand in the absence of growth hormones retained resistance. Changes in resistance persisted for three generations. It is postulated that the exogenous hormonal constituents of the culture medium are modifying the expression of genetic resistance.

  19. [In vitro regeneration and applications using vegetable cell and tissue culture].

    PubMed

    Jordán, M

    1990-10-01

    Plant cells by means of their totipotency and aided by in vitro culture techniques can be induced to perform morphogenesis leading to somatic embryoids and massive clonal multiplication; microspores or pollen can be triggered to recover haploid plants, then characters expressed via haploidy can be selected and fixed. Protoplasts from different species can lead to recombinations. We report here work done on Carica pubescens, where somatic embryoids were obtained from cells; in Prunus avium androgenesis leading to pollen calli was triggered, while plants were recovered from Nicotiana tabacum anthers. Fusion products were obtained using C. pubescens and C. papaya protoplasts, leading up to calli and shoots.

  20. Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture.

    PubMed

    Isachenko, Vladimir; Orth, Ingrid; Isachenko, Evgenia; Mallmann, Peter; Peters, Doris; Schmidt, Torsten; Morgenstern, Bernd; Foth, Dolores; Hanstein, Bettina; Rahimi, Gohar

    2013-06-01

    To achieve optimal and uniform outcomes, slow cooling protocols for human ovarian tissues generally initiate ice formation at high sub-zero temperatures (-6 to -9 °C). The aim of the study was to investigate the function of ovarian tissue that had unintentionally self seeded at -20 °C during the freezing step, by examining its development following chicken embryonic chorioallantoic membrane (CAM) grafting and after transplantation back to the patient. Ovarian tissue was frozen in 6% (v/v) dimethyl sulfoxide, 6% (v/v) ethylene glycol and 0.15M sucrose which had self-seeded at -20 °C. Five years after cryopreservation, 8 pieces were thawed and transplanted back to the patient. Two small (1 × 2 × 1 mm) pieces of this thawed tissue were cultured in a CAM-system for 5 days to assess the tissue viability. The autografted ovarian tissue re-established spontaneous menstrual bleeding within five months and raised serum 17-β Estradiol from 19 to 330 pg/ml. Ultrasound revealed a dominant follicle at the site of the transplanted tissue in the follicular phase after the menstrual bleed. Analysis of the CAM cultured tissue established that 88% of the primordial follicles are degenerated and there was limited in growth of blood vessels. In conclusion, in spite of the damage caused by the cryopreservation with spontaneous ice-formation the viability could be confirmed by CAM culture and the restoration of ovarian function after auto-transplantation.

  1. Cell suspensions, cell cultures, and tissue slices--important metabolic in vitro systems.

    PubMed

    Cervenková, K; Belejova, M; Veselý, J; Chmela, Z; Rypka, M; Ulrichová, J; Modrianský, M; Maurel, P

    2001-12-01

    In vitro subcellular and cellular systems have important and irreplaceable roles in the metabolic investigations that precede the development of new potential drugs. Of these model systems, tissue slices are probably the nearest to in vivo conditions. From the experimental and complexity points of view, perfused organs lie midway between tissue slices and whole organism. Preparation and working with liver slices is quick and easy, and, excess material can be cryopreserved and stored untill the next experiment. Slices can be prepared from a wide variety of organs and it is possible to co-incubate them. Another important feature is the possibility of interspecies comparison of slices. Different experiments can be run both in the short-term as well as long-term incubations. Each in vitro system has an important place for example, in the development of new medicaments. It is therefore important to compare and supplement experimental results from different in vitro systems when extrapolating to in vivo situations is done.

  2. Autologous fibrin scaffolds cultured dermal fibroblasts and enriched with encapsulated bFGF for tissue engineering.

    PubMed

    de la Puente, Pilar; Ludeña, Dolores; Fernández, Ana; Aranda, Jose L; Varela, Gonzalo; Iglesias, Javier

    2011-12-15

    Autologous fibrin scaffolds (AFSs) enriched with cells and specific growth factors represent a promising biocompatible scaffold for tissue engineering. Here, we analyzed the in vitro behavior of dermal fibroblasts (DFs) (cellular attachment, distribution, viability and proliferation, histological and immunohistochemical changes), comparing AFS with and without alginate microcapsules loaded with basic fibroblast growth factor (bFGF), to validate our scaffold in a future animal model in vivo. In all cases, DFs showed good adhesion and normal distribution, while in scaffolds with bFGF at 14 days, the cell counts detected in proliferation and viability assays were greatly improved, as was the proliferative state, and there was a decrease in muscle specific actin expression and collagen synthesis in comparison with the scaffolds without bFGF. In addition, the use of plasma without fibrinogen concentration methods, together with the maximum controlled release of bFGF at 14 days, favored cell proliferation. To conclude, we have been able to create an AFS enriched with fully functional DFs and release-controlled bFGF that could be used in multiple applications for tissue engineering.

  3. 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.

  4. Oxidative stress and glutathione response in tissue cultures from persons with major depression.

    PubMed

    Gibson, Sara A; Korade, Željka; Shelton, Richard C

    2012-10-01

    There is evidence that major depressive disorder (MDD) is associated with increased peripheral markers of oxidative stress. To explore oxidation and antioxidant response in MDD, we assayed human dermal fibroblast cultures derived from skin biopsies of age-, race-, and sex-matched individuals in depressed and normal control groups (n = 16 each group), cultured in glucose and galactose conditions, for relative protein carbonylation (a measure of oxidative stress), glutathione reductase (GR) expression, and total glutathione concentration. In control-group fibroblasts, galactose induced a significant increase from the glucose condition in both protein carbonylation and GR. The cells from the MDD group showed total protein carbonylation and GR expression in the glucose condition that was significantly higher than control cells in glucose and equivalent to controls in galactose. There was a small decrease in protein carbonylation in MDD cells from glucose to galactose and no significant change in GR. There was no difference in total glutathione among any of the groups. Increased protein carbonylation and GR expression, cellular responses to oxidative stress induced by galactose in control fibroblasts, are present in fibroblasts derived from MDD patients and are not explainable by reduced GR or total glutathione in the depressed patients. These studies support the role of oxidative stress in the pathophysiology of MDD. Further confirmation of these findings could lead to the development of novel antioxidant approaches for the treatment of depression.

  5. EFFECTS OF CALCIUM ION CONCENTRATION ON THE DEGENERATION OF AMPUTATED AXONS IN TISSUE CULTURE

    PubMed Central

    Schlaepfer, W. W.; Bunge, R. P.

    1973-01-01

    Light and electron microscope studies were conducted on the nature of the degenerative changes in amputated nerve fibers of cultured rat sensory ganglia and on the effects of media with differing calcium concentrations upon these changes. With glucose-enriched Eagle's media (MEM) containing 1.6 mM calcium, the amputated myelinated and unmyelinated axons undergo a progressive granular disintegration of their axoplasm with collapse and fragmentation of myelin sheaths between 6 and 24 h after transection. With MEM containing only 25–50 µM calcium, the granular axoplasmic degeneration does not occur in transected fibers and they retain their longitudinal continuity and segmental myelin ensheathment for at least 48 h. Addition of 6 mM EGTA to MEM (reducing the estimated Ca++ below 0.3 µM) results in the structural preservation of both microtubules and neurofilaments within transected axons. A transient focal swelling of amputated axons occurs, however, in cultures with normal and reduced calcium. These observations suggest that an alteration in the permeability of the axolemma is a crucial initiating event leading to axonal degenerative changes distal to nerve transection. The loss of microtubules and neurofilaments and the associated granular alterations of the axoplasm in transected fibers appears to result from the influx of calcium into the axoplasm. PMID:4805010

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

    PubMed

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

    2014-09-01

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

  7. Allelic loss of chromosome 8p22 loci in a primary tissue culture from a patient with prostate cancer

    SciTech Connect

    Godec, C.J.; Macera, M.J.; Szabo, P. |

    1994-09-01

    It is presently estimated that over 11 million men in the United States alone have histological prostate cancer. Approximately 200,000 new cases will be diagnosed in 1994 with 36,000 deaths attributed to the most common cancer in U.S. men. Although the incidence of this disease is so high, little is known about the molecular etiology of this cancer. Inactivation of tumor suppressor genes Rb and p53 have been observed at a low frequency in this disease, although allelic loss on chromosomes 8, 10 and 16 suggest possible unidentified tumor suppression genes may be more directly associated with this cancer. It was recently shown that allelic loss of D8S163, located at 8q22 {r_arrow} pter, was seen in a large percentage of primary prostate cancers. Our patient underwent a radical prostatectomy for clinically localized prostate cancer. A portion of the tissue was minced, treated with collagenase and a fibroblast-like culture was established. DNA was obtained from the culture and peripheral blood of the patient. Probe K5R2 (American Type Culture Collection) specific for loci D8S163 was hybridized to Southern blots of TagI-digested DNA. Two alleles at 3.3 Kb and 1.9 Kb were seen in the peripheral blood, while only the 1.9 Kb fragment was seen in the DNA from the culture. Loss of the allele at 3.3 Kb resembles results obtained from primary tumors, suggesting that the established line may be derived from the tumor.

  8. 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

  9. 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.

  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.

  11. Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction.

    PubMed Central

    Homberger, F R; Smith, A L; Barthold, S W

    1991-01-01

    A polymerase chain reaction (PCR) method was developed for the detection of rodent coronaviruses in biological material by using reverse transcriptase and two primers which flanked an M gene sequence of 375 bp. PCR detected all of 11 different strains of mouse hepatitis virus (MHV) as well as rat sialodacryoadenitis virus but not bovine coronavirus or human coronavirus strains OC43 and 229E. The M gene sequences of bovine coronavirus and human coronavirus OC43 are homologous to that of MHV, but minor differences exist in the primer regions, preventing annealing of the primers. For detecting MHV-Y in tissue samples, PCR was faster than and at least as sensitive as either of the two bioassays (infant mouse bioassay and mouse antibody production test) currently used for MHV diagnostic purposes. Images PMID:1661745

  12. 3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.

    PubMed

    Giselbrecht, S; Gietzelt, T; Gottwald, E; Trautmann, C; Truckenmüller, R; Weibezahn, K F; Welle, A

    2006-09-01

    We describe a new technology based on thermoforming as a microfabrication process. It significantly enhances the tailoring of polymers for three dimensional tissue engineering purposes since for the first time highly resolved surface and bulk modifications prior to a microstructuring process can be realised. In contrast to typical micro moulding techniques, the melting phase is avoided and thus allows the forming of pre-processed polymer films. The polymer is formed in a thermoelastic state without loss of material coherence. Therefore, previously generated modifications can be preserved. To prove the feasibility of our newly developed technique, so called SMART = Substrate Modification And Replication by Thermoforming, polymer films treated by various polymer modification methods, like UV-based patterned films, and films modified by the bombardment with energetic heavy ions, were post-processed by microthermoforming. The preservation of locally applied specific surface and bulk features was demonstrated e.g. by the selective adhesion of cells to patterned microcavity walls.

  13. Breeding in peach, cherry and plum: from a tissue culture, genetic, transcriptomic and genomic perspective.

    PubMed

    Carrasco, Basilio; Meisel, Lee; Gebauer, Marlene; Garcia-Gonzales, Rolando; Silva, Herman

    2013-01-01

    This review is an overview of traditional and modern breeding methodologies being used to develop new Prunus cultivars (stone fruits) with major emphasis on peach, sweet cherry and Japanese plum. To this end, common breeding tools used to produce seedlings, including in vitro culture tools, are discussed. Additionally, the mechanisms of inheritance of many important agronomical traits are described. Recent advances in stone fruit transcriptomics and genomic resources are providing an understanding of the molecular basis of phenotypic variability as well as the identification of allelic variants and molecular markers. These have potential applications for understanding the genetic diversity of the Prunus species, molecular marker-assisted selection and transgenesis. Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNPs) molecular markers are described as useful tools to describe genetic diversity in peach, sweet cherry and Japanese plum. Additionally, the recently sequenced peach genome and the public release of the sweet cherry genome are discussed in terms of their applicability to breeding programs.

  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. Comparative effects of posterior eye cup tissues from myopic and hyperopic chick eyes on cultured scleral fibroblasts.

    PubMed

    Christian, Parul G; Harkin, Damien G; Rayner, Cassie; Schmid, Katrina L

    2013-02-01

    The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5-8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA

  16. 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.

  17. Cytogenetic studies of a male with sporadic intestinal lymphangiectasia: 45,X/46,XY mosaicism with pseudo- and hyperdiploid subpopulations in cultured tissues.

    PubMed

    White, B J; Crandall, C; Flier, J S; Raveché, E S; Tjio, J H

    1979-01-01

    45,X/46,XY mosaicism was found in peripheral blood, bone marrow, and tissue cultures of an adult male with intestinal lymphangiectasia (IL). Turner phenotype was not present; his meiotic metaphase analysis was normak, and his dermatoglyphics resembled those of his family. Ten separate tissue culture lines from three biopsies of skin and thyroid gland contained 45,X cells (14.8 to 78.3%). Autosomal aneuploidy, resulting in pseudo- or hyperdiploidy, was also present in 4.3 to 41.6% of the cells. A hyperdiploid clone with a 47,X,+10,+18 karyotype was found in 22.6% of cells in one line. A second hyperdiploid clone with a 48,X,+2,+18,+18 karyotype occurred in 7.6% of cells from another line containing a total of 41.6% pseudo- and hyperdiploid cells. Such clonal abnormalities were not typical of tissue cultures from other patients done in our laboratory. Growth of our patient's tissue cultures was subnormal, and none proliferated beyond the fourth subculture. The significance of this observation remains to be determined. Our results do not allow us to conclude whether our patient's mosaicism of somatic tissues arose during embryogenesis, or whether it originated post-natally. The secondary immunodeficiency which occurs in IL may explain persistence of cells with unusual combinations of autosomal aneuploidy in our patient's tissues.

  18. A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture.

    PubMed

    Wagner, Ilka; Materne, Eva-Maria; Brincker, Sven; Süssbier, Ute; Frädrich, Caroline; Busek, Mathias; Sonntag, Frank; Sakharov, Dmitry A; Trushkin, Evgeny V; Tonevitsky, Alexander G; Lauster, Roland; Marx, Uwe

    2013-09-21

    Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a multi-organ-chip capable of maintaining 3D tissues derived from cell lines, primary cells and biopsies of various human organs. We designed a multi-organ-chip with co-cultures of human artificial liver microtissues and skin biopsies, each a (1)/100,000 of the biomass of their original human organ counterparts, and have successfully proven its long-term performance. The system supports two different culture modes: i) tissue exposed to the fluid flow, or ii) tissue shielded from the underlying fluid flow by standard Transwell® cultures. Crosstalk between the two tissues was observed in 14-day co-cultures exposed to fluid flow. Applying the same culture mode, liver microtissues showed sensitivity at different molecular levels to the toxic substance troglitazone during a 6-day exposure. Finally, an astonishingly stable long-term performance of the Transwell®-based co-cultures could be observed over a 28-day period. This mode facilitates exposure of skin at the air-liquid interface. Thus, we provide here a potential new tool for systemic substance testing.

  19. Primary tissue culture of spontaneously regressing flat warts. In vitro attack by mononuclear cells against wart-derived epidermal cells.

    PubMed

    Tagami, H; Oku, T; Iwatsuki, K

    1985-05-15

    Although tumors may be resolved due to host immune response, it is difficult to obtain direct evidence of this in man. Numerous flat warts, human papilloma virus type 3-induced papillomas, disappear systemically and simultaneously after showing inflammatory changes. Histologically, there is a dense cellular infiltration composed of lymphocytes and mononuclear phagocytes as identified by alpha-naphthyl acetate esterase staining in situ, the former being predominant in most cases. The primary tissue culture of such inflamed flat warts from ten cases revealed a proliferation of wart-derived keratinocytes as is the case with ordinary flat warts. However, in nine of the ten cases, massive mononuclear cells, most of which were T-lymphocytes, migrated out of the explants and began to attack these keratinocytes, inducing degenerative changes. These findings indicate that cell-mediated tumor cell destruction rather than antiviral reaction induces systemic spontaneous regression of multiple papillomas in man.

  20. Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.

    PubMed

    Dhimolea, Eugen; Soto, Ana M; Sonnenschein, Carlos

    2012-11-01

    Collagen-based gels have been widely used to determine the factors that regulate branching morphogenesis in the mammary gland. The patterns of biomechanical gradients and collagen reorganization influence the shape and orientation of epithelial structures in three-dimensional (3D) conditions. We explored in greater detail whether collagen type I fibers with distinct biomechanical and fiber-assembling properties, isolated from either bovine or rat tail tendon, differentially affected the epithelial phenotype in a tissue culture model of the human breast. Rat tail collagen fibers were densely packed into significantly longer and thicker bundles compared to those of the bovine type (average fascicle length 7.35 and 2.29 μm, respectively; p = 0.0001), indicating increased fiber alignment and biomechanical enablement in the former. MCF10A epithelial cells formed elaborated branched tubular structures in bovine but only nonbranched ducts and acini in rat tail collagen matrices. Ductal branching in bovine collagen was associated with interactions between neighboring structures mediated through packed collagen fibers; these fiber-mediated interactions were absent in rat tail collagen gels. Normal breast fibroblasts increased the final size and number of ducts only in rat tail collagen gels while not affecting branching. Our results suggest that the species of origin of collagen used in organotypic cultures may influence epithelial differentiation into alveolar or ductal structures and the patterns of epithelial branching. These observations underscore the importance of considering the species of origin and fiber alignment properties of collagen when engineering branching organs in 3D matrices and interpreting their role in the tissue phenotype.

  1. Evaluation of microorganisms cultured from injured and repressed tissue regeneration sites in endangered giant aquatic Ozark Hellbender salamanders.

    PubMed

    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

  2. Biochemical response of hybrid black poplar tissue culture (Populus × canadensis) on water stress.

    PubMed

    Popović, B M; Štajner, D; Ždero-Pavlović, R; Tari, I; Csiszár, J; Gallé, Á; Poór, P; Galović, V; Trudić, B; Orlović, S

    2017-02-27

    In this study, poplar tissue culture (hybrid black poplar, M1 genotype) was subjected to water stress influenced by polyethyleneglycol 6000 (100 and 200 mOsm PEG 6000). The aim of the research was to investigate the biochemical response of poplar tissue culture on water deficit regime. Antioxidant status was analyzed including antioxidant enzymes, superoxide-dismutase (SOD), catalase (CAT), guiacol-peroxidase (GPx), glutathione-peroxidase (GSH-Px), glutathione-reductase, reduced glutathione, total phenol content, Ferric reducing antioxidant power and DPPH radical antioxidant power. Polyphenol oxidase and phenylalanine-ammonium-lyase were determined as enzymatic markers of polyphenol metabolism. Among oxidative stress parameters lipid peroxidation, carbonyl-proteins, hydrogen-peroxide, reactive oxygen species, nitric-oxide and peroxynitrite were determined. Proline, proline-dehydrogenase and glycinebetaine were measured also as parameters of water stress. Cell viability is finally determined as a biological indicator of osmotic stress. It was found that water stress induced reactive oxygen and nitrogen species and lipid peroxidation in leaves of hybrid black poplar and reduced cell viability. Antioxidant enzymes including SOD, GPx, CAT and GSH-Px were induced but total phenol content and antioxidant capacity were reduced by PEG 6000 mediated osmotic stress. The highest biochemical response and adaptive reaction was the increase of proline and GB especially by 200 mOsm PEG. While long term molecular analysis will be necessary to fully address the poplar potentials for water stress adaptation, our results on hybrid black poplar suggest that glycine-betaine, proline and PDH enzyme might be the most important markers of poplar on water stress and that future efforts should be focused on these markers and strategies to enhance their concentration in poplar.

  3. 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

    2017-02-01

    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.

  4. Micro-perfusion for cardiac tissue engineering: development of a bench-top system for the culture of primary cardiac cells.

    PubMed

    Khait, Luda; Hecker, Louise; Radnoti, Desmond; Birla, Ravi K

    2008-05-01

    Tissue-engineered constructs have high metabolic requirements during in vitro culture necessitating the development of micro-perfusion systems to maintain high functional performance. In this study, we describe the design, fabrication, and testing of a novel micro-perfusion system to support the culture of primary cardiac cells. Our system consists of a micro-incubator with independent stages for 35-mm tissue culture plates with inflow/outflow manifolds for fluid delivery and aspiration. A peristaltic pump is utilized for fluid delivery and vacuum for fluid aspiration. Oxygen saturation, pH, and temperature are regulated for the media while temperature is regulated within the micro-incubator, fluid reservoir, and oxygenation chamber. Validation of the perfusion system was carried out using primary cardiac myocytes, isolated from 2- to 3-day-old neonatal rat hearts, plated on collagen-coated tissue culture plates. Two million cells/plate were used and the perfusion system was run for 1 h (without the need for a cell culture incubator) while controls were maintained in a standard cell culture incubator. We evaluated the cell viability, cell adhesion, total protein, total RNA, and changes in the expression of SERCA2 and phospholamban using RT-PCR, with N = 6 for each group. We found that there was no significant change in any variable during the 1-h run in the perfusion system. These studies served to demonstrate the compatibility of the perfusion system to support short-term culture of primary cardiac cells.

  5. Comprehensive characterization of chondrocyte cultures in plasma and whole blood biomatrices for cartilage tissue engineering.

    PubMed

    Schulz, Ronny M; Haberhauer, Marcus; Zernia, Göran; Pösel, Claudia; Thümmler, Christian; Somerson, Jeremy S; Huster, Daniel

    2014-07-01

    Many synthetic polymers and biomaterials have been used as matrices for 3D chondrocyte seeding and transplantation in the field of cartilage tissue engineering. To develop a fully autologous carrier for chondrocyte cultivation, we examined the feasibility of allogeneic plasma and whole blood-based matrices and compared them to agarose constructs. Primary articular chondrocytes isolated from 12-month-old pigs were embedded into agarose, plasma and whole blood matrices and cultivated under static-free swelling conditions for up to four weeks. To evaluate the quality of the synthesized extracellular matrix (ECM), constructs were subjected to weekly examinations using histological staining, spectrophotometry, immunohistochemistry and biochemical analysis. In addition, gene expression of cartilage-specific markers such as aggrecan, Sox9 and collagen types I, II and X was determined by RT-PCR. Chondrocyte morphology was assessed via scanning electron microscopy and viability staining, including proliferation and apoptosis assays. Finally, (13)  C NMR spectroscopy provided further evidence of synthesis of ECM components. It was shown that chondrocyte cultivation in allogeneic plasma and whole-blood matrices promoted sufficient chondrocyte viability and differentiation behaviour, resulting in neo-formation of a hyaline-like cartilage matrix.

  6. Biological activities of indoleacetylamino acids and their use as auxins in tissue culture

    SciTech Connect

    Hangarter, R.P.; Peterson, M.D.; Good, N.E.

    1980-05-01

    The auxin activities of a number of indoleacetylamino acid conjugates have been determined in three test systems: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-L-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetlyglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-L-alanine and indoleacetylglycine. The other conjugates inhibit shoot formatin weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-L-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.

  7. Study of sugar beet cyst nematode life cycle using plant tissue culture method.

    PubMed

    Shadmehr, Atena; Nouroozi, Payman; Garousi, Ghasemali; Ahmadi, Ali-Reza

    2007-09-01

    After optimization of sterilizing of cyst and larva second stage of Heterodera schachtii, possibility of using nematode on seedlings of sugar beet (Beta vulgaris L.) in in vitro conditions were studied using sterilized larvae of beet cyst nematode. For this purpose, non sterile cysts were extracted from infected soil and hatched into zinc chloride solution with concentration of 0.5 g L(-1). Then, for preparation of sterile second stage larvae, several sterilizing treatments were used. Mean comparisons were performed between sterilized live larvae number by Duncan's method. Results showed that 70% ethanol for 1 min followed by 2.5% hypochlorite sodium for 5 minutes and 0.1% hypochlorite sodium for 20 min were best treatments for disinfecting cysts and larvae, respectively. In parallel, two nematode susceptible sugar beet varieties were applied to produce seedlings in in vitro culture. PG(oB) medium containing different hormonal compositions was used for producing of hairy roots and inoculation of seedling with sterilized larvae. After nematode inoculation tests, daily observations were done by counting cysts and stained roots and larvae under stereomicroscope. Between 5-12 cysts formed on the roots of each seedling from two varieties 40 days after inoculation. As a result, it seems that this technique can be used for sugar beet germplasm evaluation to screen nematode resistant genotypes in in vitro controlled condition.

  8. Biological activities of indoleacetylamino acids and their use as auxins in tissue culture.

    PubMed

    Hangarter, R P; Peterson, M D; Good, N E

    1980-05-01

    THE AUXIN ACTIVITIES OF A NUMBER OF INDOLEACETYLAMINO ACID CONJUGATES HAVE BEEN DETERMINED IN THREE TEST SYSTEMS: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-l-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-l-alanine and indoleacetylglycine. The other conjugates inhibit shoot formation weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-l-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.

  9. Biological Activities of Indoleacetylamino Acids and Their Use as Auxins in Tissue Culture 1

    PubMed Central

    Hangarter, Roger P.; Peterson, Michael D.; Good, Norman E.

    1980-01-01

    The auxin activities of a number of indoleacetylamino acid conjugates have been determined in three test systems: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-l-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-l-alanine and indoleacetylglycine. The other conjugates inhibit shoot formation weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-l-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid. Images PMID:16661279

  10. Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

    NASA Astrophysics Data System (ADS)

    Wohlleben, Wendel; Kolle, Susanne N.; Hasenkamp, Laura-Carolin; Böser, Alexander; Vogel, Sandra; von Vacano, Bernhard; van Ravenzwaay, Ben; Landsiedel, Robert

    2011-07-01

    We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

  11. Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration.

    PubMed

    Lee, Jung-Seok; Park, Jung-Chul; Kim, Tae-Wan; Jung, Byung-Joo; Lee, Youngseok; Shim, Eun-Kyung; Park, Soyon; Choi, Eun-Young; Cho, Kyoo-Sung; Kim, Chang-Sung

    2015-09-01

    Human bone marrow mesenchymal stem cells (hBMSCs) were isolated from bone marrow of the vertebral body. The hBMSCs were cultured under either hypoxic (1% O2) or normoxic (21% O2; control) conditions and the characteristics as mesenchymal stem cells were compared. Results revealed that hypoxia reduced proliferative potential and colony-forming efficiency of hBMSCs, and significantly enhanced osteogenic and chondrogenic differentiation. The hBMSCs enhanced the regenerative potential of bone in vivo. In vitro synthesis of soluble and insoluble collagen was significantly increased in the hypoxic condition. In vivo collagen tissue regeneration was also enhanced under the hypoxic condition, with concomitant increased expressions of various subtypes of collagen and lysyl-oxidase family mRNA. MicroRNA assays revealed that miR-155-5p, which negatively regulates HIF-1α, was significantly highly expressed. These observations demonstrate that hBMSCs obtained from human vertebrae exhibit altered characteristics under hypoxic conditions, and each factor contributing to hBMSC-mediated tissue healing should be evaluated with the goal of allowing their clinical application.

  12. Mechanical influence of tissue culture plates and extracellular matrix on mesenchymal stem cell behavior: A topical review.

    PubMed

    Tatullo, Marco; Marrelli, Massimo; Falisi, Giovanni; Rastelli, Claudio; Palmieri, Francesca; Gargari, Marco; Zavan, Barbara; Paduano, Francesco; Benagiano, Vincenzo

    2016-03-01

    Tissue engineering applications need a continuous development of new biomaterials able to generate an ideal cell-extracellular matrix interaction. The stem cell fate is regulated by several factors, such as growth factors or transcription factors. The most recent literature has reported several publications able to demonstrate that environmental factors also contribute to the regulation of stem cell behavior, leading to the opinion that the environment plays the major role in the cell differentiation.The interaction between mesenchymal stem cells (MSCs) and extracellular environment has been widely described, and it has a crucial role in regulating the cell phenotype. In our laboratory (Tecnologica Research Institute, Crotone, Italy), we have recently studied how several physical factors influence the distribution and the morphology of MSCs isolated from dental pulp, and how they are able to regulate stem cell differentiation. Mechanical and geometrical factors are only a small part of the environmental factors able to influence stem cell behavior, however, this influence should be properly known: in fact, this assumption must be clearly considered during those studies involving MSCs; furthermore, these interactions should be considered as an important bias that involves an high number of studies on the MSCs, since in worldwide laboratories the scientists mostly use tissue culture plates for their experiments.

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

    SciTech Connect

    Funk, C.; Croteau, R. )

    1993-04-01

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

  14. Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).

    PubMed Central

    Funk, C.; Croteau, R.

    1993-01-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. PMID:12231778

  15. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing.

    PubMed

    Moffitt, Jeffrey R; Hao, Junjie; Bambah-Mukku, Dhananjay; Lu, Tian; Dulac, Catherine; Zhuang, Xiaowei

    2016-12-13

    Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background-from off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.

  16. Expression of miR-15/107 Family MicroRNAs in Human Tissues and Cultured Rat Brain Cells

    PubMed Central

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

    2014-01-01

    The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs), sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively). In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS). In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs). Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes. PMID:24480177

  17. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

    PubMed Central

    Moffitt, Jeffrey R.; Hao, Junjie; Bambah-Mukku, Dhananjay; Lu, Tian; Dulac, Catherine

    2016-01-01

    Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background—from off-target binding of FISH probes and cellular autofluorescence—can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues. PMID:27911841

  18. Synthesis and evaluation of bioceramics for orthopedics and tissue culture applications

    NASA Astrophysics Data System (ADS)

    Demirkiran, Hande

    Hydroxyapatite is the most well known phosphate in the biologically active phosphate ceramic family by virtue of its similarity to natural bone mineral. Among all bioglass compositions BioglassRTM45S5 is one of the most bioactive glasses. This study initially started by adding different amounts (1, 2.5, 5, 10, and 25 wt.%) of BioglassRTM45S5 to synthetic hydroxyapatite in order to improve the bioactivity of these bioceramics. The chemistries formed by sintering and their effect on different material properties including bioactivity were identified by using various techniques, such as powder and thin film x-ray diffraction, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, X-ray absorption near edge spectroscopy, compression test, and nano indentation. All the results demonstrated that 10 and 25 wt.% BioglassRTM45S5 addition to hydroxyapatite and sintering at 1200°C for 4 hours yield new compositions with main Ca 5(PO4)2SiO4 and Na3Ca 6(PO4)5 crystalline phases dispersed in silicate glassy matrices, respectively. In addition, in vitro bioactivity tests such as bone like apatite formation in simulated body fluid and bone marrow stromal cell culture have shown that the crystalline and amorphous phases have an important role on improving bioactivity of these bioceramic compositions. Besides, compression test and nano indentation has given important information on compression strength and nano structure properties of these newly composed bioceramic materials and the bone like apatite layers formed on them, respectively. Finally, the effect of silicate addition on both formation and bioactivity of Na3Ca6(PO4)5 bioceramics were shown. These findings and different techniques used assisted to develop a phenomenological approach to demonstrate how the novel bioceramic compositions were composed and aid improving bioactivity of known bioceramic materials.

  19. Erythropoietin triggers a burst of GATA-1 in normal human erythroid cells differentiating in tissue culture.

    PubMed Central

    Dalyot, N; Fibach, E; Ronchi, A; Rachmilewitz, E A; Ottolenghi, S; Oppenheim, A

    1993-01-01

    GATA-1 is a central transcription-activator of erythroid differentiation. In the present work we have studied the kinetics of its expression and activity during development of normal human erythroid progenitors, grown in primary cultures. In response to the addition of erythropoietin (Epo), the cells undergo proliferation and differentiation in a synchronized fashion. This recently developed experimental system allows biochemical dissection of erythroid differentiation in a physiological meaningful environment. No DNA-binding activity of GATA-1 could be detected before the addition of Epo, although a very low level of mRNA was observed. Following Epo addition there was a sharp parallel rise in both mRNA and DNA-binding activity, consistent with positive autoregulation of the GATA-1 gene. After reaching a peak on day 7-9, both mRNA and protein activity decreased. The binding activity of the ubiquitous factor SP1 showed a biphasic pattern; its second peak usually coincided with the GATA-1 peak, suggesting that SP1 also plays a specific role in erythroid maturation. The highest activity of GATA-1 per erythroid cell was found on day 6-8, immediately preceding the major rise in globin gene mRNA and in the number of hemoglobinized cells. The results imply that a high level of GATA-1 activity is necessary for globin gene expression and erythroid maturation, suggesting that a requirement for a threshold concentration of GATA-1 is part of the mechanism that determines the final steps of erythroid maturation. Images PMID:8371977

  20. Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

    PubMed

    Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

    2017-04-01

    The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

  1. The pregnant ferret as a model for studying the congenital effects of influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo.

    PubMed

    Sweet, C; Toms, G L; Smith, H

    1977-04-01

    Organ cultures of ferret foetal tissues showed a similar pattern of susceptibility to influenza virus to that already observed for human foetal tissues (Rosztoczy et al., 1975); respiratory, alimentary and urogenital tissues supported the replication of influenza virus but nervous and lymphopoietic tissues (those which, in man, are associated with foetal or postnatal abnormalities) were insusceptible. In contrast to corresponding human tissues, ferret foetal placenta and amnion readily supported viral replication although both human and ferret umbilical cord were susceptible. In limited experiments, neither the membranes nor the susceptible foetal tissues became infected after intranasal inoculation of pregnant ferrets of various gestational ages. However, after intracardial inoculation of pregnant ferrets with high titre virus (ca 10(9) EBID50) virus was isolated from both foetal membranes and foetuses. The membranes became infected at early, middle and late gestation, but virus appeared to cross the placental barrier to infect foetal tissues only in late gestation. At this stage virus could be isolated not only from those foetal tissues (respiratory, alimentary and urogenital) susceptible in organ culture, but also in small amounts from tissues which were insusceptible in organ culture (heart, lymphopoietic and nervous tissue). Virus was also isolated from foetal membranes and foetuses of late gestation ferrets following intracardial inoculation with a one hundred-fold lower dose of virus which, unlike the higher dose, did not induce a maternal febrile response. The pregnant ferret appears to be a suitable model for investigating the effects on development of foetal infection with influenza virus but it may have disadvantages with regard to the nature and strength of the placental barrier.

  2. Effects of cell culture techniques on gene expression and cholesterol efflux in primary bovine mammary epithelial cells derived from milk and tissue.

    PubMed

    Sorg, D; Potzel, A; Beck, M; Meyer, H H D; Viturro, E; Kliem, H

    2012-10-01

    Primary bovine mammary epithelial cells (pbMEC) are often used in cell culture to study metabolic and inflammatory processes in the udder of dairy cows. The most common source is udder tissue from biopsy or after slaughter. However, it is also possible to culture them from milk, which is non-invasive, repeatable and yields less contamination with fibroblasts. Generally, not much is known about the influence of cell origin and cell culture techniques such as cryopreservation on pbMEC functionality. Cells were extracted from milk and udder tissue to evaluate if milk-derived pbMEC are a suitable alternative to tissue-derived pbMEC and to test what influence cryopreservation has. The cells were cultivated for three passages and stored in liquid nitrogen. The relative gene expression of the five target genes kappa-casein, lingual antimicrobial peptide (LAP), lactoferrin, lysozyme (LYZ1) and the prolactin receptor normalised with keratin 8 showed a tendency to decrease in the tissue cultures, but not in the milk-derived cultures, suggesting a greater influence of the cultivation process on tissue-derived cells, freezing lowered expression levels in both cultures. Overall expression of LAP and LYZ1 tended to be higher in milk cells. Cholesterol efflux was measured to compare passages one to seven in milk-derived cells. Passage number did not alter the efflux rate (p ≤ 0.05). We showed for the first time that the extraction of pbMEC from milk can be a suitable alternative to tissue extraction.

  3. A three-dimensional tissue culture model for the study of attach and efface lesion formation by enteropathogenic and enterohaemorrhagic Escherichia coli.

    PubMed

    Carvalho, Humberto M; Teel, Louise D; Goping, Gertrud; O'Brien, Alison D

    2005-12-01

    We sought to develop a practical and representative model to study the interactions of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) with human intestinal tissue. For this purpose, human intestinal epithelial HCT-8 cells were cultured under low-shear microgravity conditions in a rotating cell culture system. After 10 days, layered cell aggregates, or 'organoids', developed. Three lines of evidence indicated that these organoids exhibited traits characteristic of normal tissue. First, the organoids expressed normal intestinal tissue markers in patterns that suggested greater cellular differentiation in the organoids than conventionally grown monolayers. Second, the organoids produced higher levels of intestinally expressed disaccharidases and alkaline phosphatase on a cell basis than did conventionally cultured monolayers. Third, HCT-8 organoid tissue developed microvilli and desmosomes characteristic of normal tissue, as revealed by electron microscopy. Because the low-shear microgravity condition is proposed by modelling studies to more closely approximate conditions in the intestinal microvilli, we also tested the impact of microgravity of bacterial growth and virulence gene expression. No influence on growth rates was observed but intimin expression by EHEC was elevated during culture in microgravity as compared with normal gravity. That the responses of HCT-8 organoids to infection with wild-type EPEC or EHEC under microgravitational conditions approximated infection of normal tissue was demonstrated by the classical appearance of the resultant attaching and effacing lesions. We concluded that the low shear microgravity environment promoted growth of intestinal cell organoids with greater differentiation than was seen in HCT-8 cells maintained in conventional tissue culture and provided a reduced gravity environment for study of bacterial-host cell interactions.

  4. Culture.

    ERIC Educational Resources Information Center

    1997

    Twelve conference papers on cultural aspects of second language instruction include: "Towards True Multiculturalism: Ideas for Teachers" (Brian McVeigh); Comparing Cultures Through Critical Thinking: Development and Interpretations of Meaningful Observations" (Laurel D. Kamada); "Authority and Individualism in Japan and the…

  5. Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor.

    PubMed

    Ishikawa, Momotaro; Sekine, Keisuke; Okamura, Ai; Zheng, Yun-wen; Ueno, Yasuharu; Koike, Naoto; Tanaka, Junzo; Taniguchi, Hideki

    2011-06-01

    Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

  6. Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

    PubMed Central

    Rotondo, Floriana; Romero, María del Mar; Ho-Palma, Ana Cecilia; Remesar, Xavier; Fernández-López, José Antonio

    2016-01-01

    Background White adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown. Experimental Design Epididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT. Conclusions The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination). These data translate (with respect to the

  7. Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

    PubMed

    Liu, Jess; Czernick, Drew; Lin, Shih-Chun; Alasmari, Abeer; Serge, Dibart; Salih, Erdjan

    2013-09-01

    Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin. In our resorption model live bone cultures were grown in the absence of ascorbate and were stimulated by parathyroid hormone (PTH) to undergo rapid osteoclast formation/differentiation with bone resorption. In this resorption model native phosvitin potently inhibited PTH-induced osteoclastic bone resorption with simultaneous new osteoid/bone formation in the absence of ascorbate (vitamin C). These surprising and critical observations were extended using the bone formation model in the absence of ascorbate and in the presence of phosvitin which supported the above results. The results were corroborated by analyses for calcium release or uptake, tartrate-resistant acid phosphatase activity (marker for osteoclasts), alkaline phosphatase activity (marker for osteoblasts), collagen and hydroxyproline composition, and histological and quantitative histomorphometric evaluations. The data revealed that the discovered bioactivity of phosvitin mirrors that of ascorbate during collagen synthesis and the formation of new osteoid/bone. Complementing those studies use of the synthetic collagen peptide analog and cultured calvarial osteoblasts in conjunction with mass spectrometric analysis provided results that augmented the bone organ culture work and confirmed the capacity of phosvitin to stimulate differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization. There are striking implications and interrelationships of this affect that relates to

  8. Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures.

    PubMed

    Inaba, Yoshimi; Zhong, Wei Qun; Zhang, Xing-Hai; Widholm, Jack M

    2007-07-01

    Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than

  9. Comparison of Genotoxic Damage in Monolayer Cell Cultures and Three-Dimensional Tissue-Like Cell Assemblies

    NASA Technical Reports Server (NTRS)

    Behravesh, E.; Emami, K.; Wu, H.; Gonda, S.

    2004-01-01

    Assessing the biological risks associated with exposure to the high-energy charged particles encountered in space is essential for the success of long-term space exploration. Although prokaryotic and eukaryotic cell models developed in our laboratory and others have advanced our understanding of many aspects of genotoxicity, in vitro models are needed to assess the risk to humans from space radiation insults. Such models must be representative of the cellular interactions present in tissues and capable of quantifying I genotoxic damage. Toward this overall goal, the objectives of this study were to examine the effect of the localized microenvironment of cells, cultured as either 2-dimensional (2D) monolayers or 3-dimensional (3D) aggregates, on the rate and type of genotoxic damage resulting from exposure to iron charged particles, a significant portion of space radiation. We used rodent transgenic cell lines containing 50-70 copies of a LacI transgene to provide the enhanced sensitivity required to quantify mutational frequency and type in the 1,100-bp LacI target as well as assessment of DNA,damage to the entire 45-kbp construct. Cultured cells were exposed to high-enerir on charged particles at Brookhaven National Laboratory s Alternating Gradient Synchrotron facility for a total dose of 0, 0.1, 0.25,0.5, 1.0, or 2.0 Gy and allowed to recover for 0, 1, or 7 days, after which mutational type and frequency were evaluated. The mutational frequency was found to be higher in 3D samples than in 2D samples at all radiation doses. Mutational frequency also was higher at 7 days after irradiation than immediately after exposure. DNA sequencing of the mutant targets revealed that deletional mutations contributed an increasingly high percentage (up to 27%) of all mutations in cells as the dose was increased from 0.5 to 2 Gy. Several mutants also showed large and complex deletions in multiple locations within the Lac1 target. However, no differences in mutational type were

  10. Periodontal ligament cells cultured under steady-flow environments demonstrate potential for use in heart valve tissue engineering.

    PubMed

    Martinez, Catalina; Rath, Sasmita; Van Gulden, Stephanie; Pelaez, Daniel; Alfonso, Abraham; Fernandez, Natasha; Kos, Lidia; Cheung, Herman; Ramaswamy, Sharan

    2013-02-01

    A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ~5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.

  11. Neuroprotection of cerebrolysin in tissue culture models of brain ischemia: post lesion application indicates a wide therapeutic window.

    PubMed

    Schauer, E; Wronski, R; Patockova, J; Moessler, H; Doppler, E; Hutter-Paier, B; Windisch, M

    2006-07-01

    All attempts to reduce neuronal damage after acute brain ischemia by the use of neuroprotective compounds have failed to prove efficacy in clinical trials so far. One of the main reasons might be the relatively narrow time window for intervention. In this study 2 different tissue culture models of ischemia, excitotoxic lesion by the use of glutamate and oxygen-glucose deprivation (OGD), were used to investigate the effects of delayed application of Cerebrolysin (Cere) on neuronal survival. This drug consists of low molecular weight peptides with neuroprotective and neurotrophic properties similar to naturally occurring growth factors. After both types of lesion, acute as well as delayed treatment with Cere resulted in a dose dependent and significant rescue of neurons. In the model of excitotoxic cell death significant drug effects were found even when the treatment started with a delay of 96 hours after addition of glutamate. In the OGD model pronounced effects were found after 48 hours delay of treatment, and even after 72 hours a small but significant rescue of neurons was detected. The neuroprotective effects of a single addition of Cerebrolysin to the culture medium resulted in significant protection until end of the experiments which was up to 2 weeks after the initial lesion. A shift of the efficacious dosages from low to high concentrations indicates that most likely active compounds are used up, indicating that multiple dosing might even increase the effect size. In conclusion the results indicate that Cere displays a relatively wide therapeutic time window which might be explained by a combination of acute neuroprotective properties and neurotrophic efficacy.

  12. Analysis of the genotoxic potential of low concentrations of Malathion on the Allium cepa cells and rat hepatoma tissue culture.

    PubMed

    Bianchi, Jaqueline; Mantovani, Mario Sérgio; Marin-Morales, Maria Aparecida

    2015-10-01

    Based on the concentration of Malathion used in the field, we evaluated the genotoxic potential of low concentrations of this insecticide on meristematic and F1 cells of Allium cepa and on rat hepatoma tissue culture (HTC cells). In the A. cepa, chromosomal aberrations (CAs), micronuclei (MN), and mitotic index (MI) were evaluated by exposing the cells at 1.5, 0.75, 0.37, and 0.18mg/mL of Malathion for 24 and 48hr of exposure and 48hr of recovery time. The results showed that all concentrations were genotoxic to A. cepa cells. However, the analysis of the MI has showed non-relevant effects. Chromosomal bridges were the CA more frequently induced, indicating the clastogenic action of Malathion. After the recovery period, the higher concentrations continued to induce genotoxic effects, unlike the observed for the lowest concentrations tested. In HTC cells, the genotoxicity of Malathion was evaluated by the MN test and the comet assay by exposing the cells at 0.09, 0.009, and 0.0009mg/5mL culture medium, for 24hr of exposure. In the comet assay, all the concentrations induced genotoxicity in the HTC cells. In the MN test, no significant induction of MN was observed. The genotoxicity induced by the low concentrations of Malathion presented in this work highlights the importance of studying the effects of low concentrations of this pesticide and demonstrates the efficiency of these two test systems for the detection of genetic damage promoted by Malathion.

  13. Improving knowledge of plant tissue culture and media formulation by neurofuzzy logic: a practical case of data mining using apricot databases.

    PubMed

    Gago, Jorge; Pérez-Tornero, Olaya; Landín, Mariana; Burgos, Lorenzo; Gallego, Pedro P

    2011-10-15

    Plant tissue growth can be regulated and controlled via culture media composition. A number of different laborious and time-consuming approaches have been used to attempt development of optimized media for a wide range of species and applications. However, plant tissue culture is a very complex task, and the identification of the influences of process factors such as mineral nutrients or plant growth regulators on a wide spectrum of growth responses cannot always well comprehended. This study employs a new approach, data mining, to uncover and integrate knowledge hidden in multiple data from plant tissue culture media formulations using apricot micropropagation databases as an example. Neurofuzzy logic technology made it possible to identify relationships among several factors (cultivars, mineral nutrients and plant growth regulators) and growth parameters (shoots number, shoots length and productivity), extracting biologically useful information from each database and combining them to create a model. The IF-THEN rule sets generated by neurofuzzy logic were completely in agreement with previous findings based on statistical analysis, but advantageously generated understandable and reusable knowledge that can be applied in future plant tissue culture media optimization.

  14. Regulation of Embryo Dormancy by Manipulation of Abscisic Acid in Kernels and Associated Cob Tissue of Zea mays L. Cultured in Vitro1

    PubMed Central

    Hole, David J.; Smith, J. D.; Cobb, B. Greg

    1989-01-01

    Sectors of Zea mays cobs, with and without kernels were cultured in vitro in the presence and absence of fluridone. Cultured kernels, cob tissue, and embryos developed similarly to those grown in the field. Abscisic acid (ABA) levels in the embryos were evaluated by enzyme-linked immunosorbant assay. ABA levels in intact embryos cultured in the presence of fluridone were extremely low and indicate an inhibition of ABA synthesis. ABA levels in isolated cob tissue indicate that ABA can be produced by cob tissue. Sections containing kernels cultured in the presence of fluridone were transferred to medium containing fluridone and ABA. Dormancy was induced in more than 50% of the kernels transferred from 13 to 15 days after pollination, but all of the kernels transferred at 16 days after pollination or later were viviparous. ABA recovered from kernels that were placed in medium containing fluridone and ABA suggest that ABA can be transported through the cob tissue into developing embryos and that ABA is required for induction of dormancy in intact embryos. PMID:16666978

  15. Analysis of gravity-induced particle motion and fluid perfusion flow in the NASA-designed rotating zero-head-space tissue culture vessel

    NASA Technical Reports Server (NTRS)

    Wolf, David A.; Schwarz, Ray P.

    1991-01-01

    The gravity induced motions, through the culture media, is calculated of living tissue segments cultured in the NASA rotating zero head space culture vessels. This is then compared with the media perfusion speed which is independent of gravity. The results may be interpreted as a change in the physical environment which will occur by operating the NASA tissue culture systems in actual microgravity (versus unit gravity). The equations governing particle motions which induce flows at the surface of tissues contain g terms. This allows calculation of the fluid flow speed, with respect to a cultured particle, as a function of the external gravitational field strength. The analysis is approached from a flow field perspective. Flow is proportional to the shear exerted on a structure which maintains position within the field. The equations are solved for the deviation of a particle from its original position in a circular streamline as a function of time. The radial deviation is important for defining the operating limits and dimensions of the vessel because of the finite radius at which particles necessarily intercept the wall. This analysis uses a rotating reference frame concept.

  16. Supportive development of functional tissues for biomedical research using the MINUSHEET® perfusion system

    PubMed Central

    2012-01-01

    Functional tissues generated under in vitro conditions are urgently needed in biomedical research. However, the engineering of tissues is rather difficult, since their development is influenced by numerous parameters. In consequence, a versatile culture system was developed to respond the unmet needs. Optimal adhesion for cells in this system is reached by the selection of individual biomaterials. To protect cells during handling and culture, the biomaterial is mounted onto a MINUSHEET® tissue carrier. While adherence of cells takes place in the static environment of a 24 well culture plate, generation of tissues is accomplished in one of several available perfusion culture containers. In the basic version a continuous flow of always fresh culture medium is provided to the developing tissue. In a gradient perfusion culture container epithelia are exposed to different fluids at the luminal and basal sides. Another special container with a transparent lid and base enables microscopic visualization of ongoing tissue development. A further container exhibits a flexible silicone lid to apply force onto the developing tissue thereby mimicking mechanical load that is required for developing connective and muscular tissue. Finally, stem/progenitor cells are kept at the interface of an artificial polyester interstitium within a perfusion culture container offering for example an optimal environment for the spatial development of renal tubules. The system presented here was evaluated by various research groups. As a result a variety of publications including most interesting applications were published. In the present paper these data were reviewed and analyzed. All of the results point out that the cell biological profile of engineered tissues can be strongly improved, when the introduced perfusion culture technique is applied in combination with specific biomaterials supporting primary adhesion of cells. PMID:23369669

  17. Characterization of a unique OpMNPV-specific early gene not required for viral infection in tissue