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Sample records for suspension culture cell

  1. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    of growth regulators were observed to be 3 × 10−6M indoleacetic acid (JAA) combined with 3 × 10−6M benzylaminopurin (BAP) or 10−6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further....... Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  2. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

    Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

    2000-01-01

    Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

  3. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...

  4. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    to affect the growth and metabolism of cultured cells, and have been studied extensively in different species in .... Specific growth rate of neem cell suspensions in altered nitrate: ammonium ratio. MS, Murashige and Skoog medium; MS medium with .... Stimulated caffeine production has been reported in Coffea arabica cell ...

  5. Embryo forming cells in carrot suspension cultures

    NARCIS (Netherlands)

    Toonen, M.A.J.

    1997-01-01


    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic

  6. Production of recombinant proteins in suspension-cultured plant cells.

    Science.gov (United States)

    Plasson, Carole; Michel, Rémy; Lienard, David; Saint-Jore-Dupas, Claude; Sourrouille, Christophe; de March, Ghislaine Grenier; Gomord, Véronique

    2009-01-01

    Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. Among various plant expression systems being explored, genetically modified suspension-cultured plant cells offer a promising system for production of biopharmaceuticals. Indeed, when compared to other plant-based production platforms that have been explored, suspension-cultured plant cells have the advantage of being totally devoid of problems associated with the vagaries of weather, pest, soil and gene flow in the environment. Because of short growth cycles, the timescale needed for the production of recombinant proteins in plant cell culture can be counted in days or weeks after transformation compared to months needed for the production in transgenic plants. Moreover, recovery and purification of recombinant proteins from plant biomass is an expensive and technically challenging business that may amount to 80-94% of the final product cost. One additional advantage of plant cell culture is that the recombinant protein fused with a signal sequence can be expressed and secreted into the culture medium, and therefore recovered and purified in the absence of large quantities of contaminating proteins. Consequently, the downstream processing of proteins extracted from plant cell culture medium is less expensive, which may/does balance the higher costs of fermentation. When needed for clinical use, recombinant proteins are easily produced in suspension-cultured plant cells under certified, controllable and sterile conditions that offer improved safety and provide advantages for good manufacturing practices and regulatory compliance. In this chapter, we present basic protocols for rapid generation of transgenic suspension-cultured cells of Nicotiana tabacum, Oriza sativa and Arabidopis

  7. In vitro plant regeneration from embryogenic cell suspension culture ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... plasts in A. adsurgens (Luo and Jia, 1998a, b). There are only few reports available on plant regeneration systems via somatic embryogenesis (Luo et al., 1999; Hou and. Jia, 2004). Here, we report a protocol for plant regenera- tion from embryogenic cell suspension culture of endemic. A. chrysochlorus.

  8. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of.

  9. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Key words: Sorghum, proteomics, callus, cell suspension cultures, total soluble protein, secretome. INTRODUCTION. Sorghum, a cereal crop native to Africa, is drought- tolerant, surviving periods of water deficit (Rosenow et al., 1983). The crop is grown in the semi-arid regions of. Africa and Asia primarily ...

  10. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of E. angustifolia was ...

  11. Elicitation of Diacetylenic Compounds in Suspension Cultured Cells of Eggplant

    Science.gov (United States)

    Imoto, Setsuko; Ohta, Yoshimoto

    1988-01-01

    Induction of stress metabolites in the suspension cultured cells of eggplant (Solanum melongena L.) was examined. When autoclaved RNase A or nigeran, both of which are nonspecific phytoalexin elicitors in bean cells, were added to the cell culture of eggplant, greatly enhanced levels of three compounds were observed. One of them was cis-pentadeca-6-ene-1,3-diyne-5,15-diol, a novel diacetylenic compound. This compound has considerable fungitoxic activity. Also identified was falcarindiol, another fungitoxic diacetylenic compound previously reported as one of the phytoalexins in infected tomato fruits and leaves. Elicited compounds preferentially accumulated in the culture medium rather than in the cells and decreased to original levels during prolonged culturing. The elicitation of these compounds was closely correlated with cellular damage in terms of the decrease of growth rate and was inhibited by 10 micromolar cycloheximide. PMID:16665862

  12. Isolation and culture of Celosia cristata L cell suspension protoplasts

    Directory of Open Access Journals (Sweden)

    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  13. ( Linum usitatissimum L. cv. Modran cell suspension culture

    Directory of Open Access Journals (Sweden)

    Aleksandra Seta-Koselska

    2018-01-01

    Full Text Available Flax ( Linum usitatissimum L. is an ancient crop that is widely cultivated as a source of oil, fiber, and bioactive compounds. Flax fiber is traditionally used in textile industry, linseed oil is processed for industrial oils, paints, varnishes and bio-petroleum. Flaxseeds are also rich in α-linolenic acid and phytochemicals such as lignans. In addition to the commercial aspects, this species has been used widely and readily in biotechnological, developmental, and plant-pathogen interaction studies. Differences in the levels of endogenous hormones in various cultivars of flax significantly affected the intensity of callogenesis and determined the type and concentration of growth regulators necessary for callus production. The aim of our investigation was to optimize the culture conditions for callus formation and cell proliferation in liquid medium of the Polish cultivar of fiber flax – Modran. In the first step, 4 combinations of phytohormones in the medium were tested to obtain established callus tissue suitable for initiation of suspension culture. Next, we investigated the effect of chosen plant growth regulators on cell divisions, fresh and dry weight, and dispersal of callus cells in liquid medium. Fast growing and friable callus was obtained in a modified MS medium supplemented with 0.5 mg/l BAP and 0.1 mg/l NAA. We determined that for the initiation of cell suspension supplementation with 0.5 mg/l BAP and 0.5 mg/l NAA is optimal. The results obtained indicated that high concentration of cytokinin (BAP in liquid medium limited cell proliferation and decreased biomass formation.

  14. The phosphatidylinositol species of suspension cultured plant cells

    International Nuclear Information System (INIS)

    Heim, S.; Wagner, K.G.

    1987-01-01

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with [ 3 H]inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different 3 H-labeled species by autoradiography. The ratio of [ 3 H]inositol incorporation into PI, PIP and PIP 2 was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound. (orig.)

  15. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...

  16. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate:ammonium showed that the ratio 4:1 revealed a profound effect, ...

  17. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-05

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.

  18. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris

    NARCIS (Netherlands)

    Koulman, A; Kubbinga, M.E.; Batterman, S; Woerdenbag, H.J.; Pras, N.; Woolley, J.G.; Quax, Wim

    2003-01-01

    In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which

  19. An efficient method for the establishment of cell suspension cultures in potato (Solanum tuberosum L.)

    International Nuclear Information System (INIS)

    Sajid, Z.A.

    2016-01-01

    Cell suspension cultures offers an In vitro system that can be used as a tool for various studies involving mutant selection, mass propagation, protoplast isolation, gene transfer and selection of cell-lines which are resistant to various biotic or abiotic stresses. Research work on the development of cell suspension cultures was carried out to establish the most efficient method in Potato (cv. Desiree). Healthy, well-proliferating tissues from different types of callus cultures (compact, friable, embryogenic or non-embryogenic) were inoculated on various media combinations, i.e., MS, MS2 or AA liquid medium containing 18.09 micro M 2, 4-D. A fixed quantity (0.5-1.0 g) of callus tissue from 60-day-old callus cultures was transferred to 10-25 ml of liquid medium in 100 ml Erlenmeyer flask. Cultures were placed on an orbital shaker and agitated at different speeds (75, 100 or 125 rpm) under 16-h photoperiod at 25 ± 2 degree C. Medium was changed after every 3 days and fractionated tissue was filtered after every 6 days through sterile mesh (100-800 micro m) to develop a cell-line by transferring resulting suspension to fresh medium under the same conditions. Results indicated that eight-week-old translucent, friable, off-white callus cultures were an excellent starting material for the initiation of homogeneous cell suspension cultures as compared to other tested sources. Of the three tested media (MS, MS2 or AA medium containing 18.09 micro M 2, 4-D), MS2 was found to be a better medium for the initiation of cell suspension cultures. Cell suspension cultures, placed in 16-h photoperiod at 25 ± 2 degree C and agitated at 120 rpm using a gyratory shaker showed excellent results. Several other factors influencing quick establishment of cell suspension cultures in this cultivar are also discussed in this communication. (author)

  20. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    International Nuclear Information System (INIS)

    Kim, Jae Sung; Lee, Young Keun; Chung, Byung Yeoup; Lee, Young Bok; Hwang Hye Yeon

    2004-10-01

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark, and was increased by adding 1 μM Cu 2+ and 100 μM methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg·L -1 and IAA 0.2 mg·L -1 in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of γ radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS (β-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs

  1. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45...

  2. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different ...

  3. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  4. The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures.

    Science.gov (United States)

    Cetin, Emine Sema; Babalik, Zehra; Hallac-Turk, Filiz; Gokturk-Baydar, Nilgun

    2014-09-23

    Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM) to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols) production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols) and dry cell weights were determined in the harvested cells. Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g), total flavanol (15.94 mg/100 g), total flavonol (14.73 mg/100 g) and trans-resveratrol (490.76 μg/100 g) were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 μg/100 g) were detected in the cell cultures collected at day 6

  5. The effects of cadmium chloride on secondary metabolite production in Vitis vinifera cv. cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Emine Sema Cetin

    2014-01-01

    Full Text Available BACKGROUND: Plant secondary metabolites are possess several biological activities such as anti-mutagenic, anti-carcinogenic, anti-aging, etc. Cell suspension culture is one of the most effective systems to produce secondary metabolites. It is possible to increase the phenolic compounds and tocopherols by using cell suspensions. Studies on tocopherols production by cell suspension cultures are seldom and generally focused on seed oil plants. Although fresh grape, grape seed, pomace and grape seed oil had tocopherols, with our best knowledge, there is no research on tocopherol accumulation in the grape cell suspension cultures. In this study, it was aimed to determine the effects of cadmium chloride treatments on secondary metabolite production in cell suspension cultures of grapevine. Cell suspensions initiated from callus belonging to petiole tissue was used as a plant material. Cadmium chloride was applied to cell suspension cultures in different concentration (1.0 mM and 1.5 mM to enhance secondary metabolite (total phenolics, total flavanols, total flavonols, trans-resveratrol, and α-, β-, γ- δ-tocopherols production. Cells were harvested at two days intervals until the 6th day of cultures. Amounts of total phenolics, total flavanols and total flavonols; trans-resveratrol and tocopherols (α-, β-, γ- and δ-tocopherols and dry cell weights were determined in the harvested cells. RESULTS: Phenolic contents were significantly affected by the sampling time and cadmium concentrations. The highest values of total phenolic (168.82 mg/100 g, total flavanol (15.94 mg/100 g, total flavonol (14.73 mg/100 g and trans-resveratrol (490.76 µg/100 g were found in cells treated with 1.0 mM CdCl2 and harvested at day 2. Contents of tocopherols in the cells cultured in the presence of 1.0 mM CdCl2 gradually increased during the culture period and the highest values of α, β and γ tocopherols (145.61, 25.52 and 18.56 µg/100 g were detected in the cell

  6. Feruloyl Oligosaccharides from Cell Walls of Suspension-Cultured Spinach Cells and Sugar Beet Pulp : STRUCTURE AND FUNCTION OF CELLS

    OpenAIRE

    Tadashi, ISHII; Forestry and Forest Products Research Institute

    1994-01-01

    Cell walls of suspension-cultured spinach cells and sugar beet pulp were separately hydrolyzed with Driselase. A feruloyl arabinobiose was isolated from both spinach cells and sugar beet. Four feruloyl oligosaccharides were obtained from sugar beet. The four oligosaccharides were characterized by NMR spectroscopy, methylation analysis and FAB-MS.

  7. Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures.

    Science.gov (United States)

    Ashmore, S E; Shapcott, A S

    1989-08-01

    Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change.

  8. Cell line selection combined with jasmonic acid elicitation enhance camptothecin production in cell suspension cultures of Ophiorrhiza mungos L.

    Science.gov (United States)

    Deepthi, S; Satheeshkumar, K

    2017-01-01

    Ophiorrhiza mungos is a herbaceous medicinal plant which contains a quinoline alkaloid, camptothecin (CPT), an anticancer compound. A high-yielding cell line, O. mungos cell line-3 (OMC3) was selected from cell suspension cultures of O. mungos using cell aggregate cloning method and established cell suspension culture. OMC3 cell suspension produced significantly high biomass (9.25 ± 1.3 g/flask fresh weight (FW)) and CPT yield (0.095 ± 0.002 mg g -1 dry weight (DW)) compared with the original cell suspension. Inoculum size of OMC3 cell suspension culture was optimised as 14 g L -1 . Media optimisation has shown that 5 % (w/v) sucrose and an increased ammonium/nitrate concentration of 40/20 mM favoured CPT production, whereas 3 % (w/v) sucrose, an ammonium/nitrate concentration of 20/40 mM and 1.25 mM of phosphate favoured biomass accumulation. Jasmonic acid, chitin and salicylic acid was used to elicit CPT production in the original cell suspension culture and achieved significantly high CPT production with jasmonic acid (JA) elicitation. Further, OMC3 cell suspension culture was elicited with JA (50 μM) and obtained 1.12 ± 0.08 mg g -1 DW CPT and 9.52 ± 1.4 g/flask FW (190.4 g L -1 FW). The combination of cell line selection and elicitation has produced 18.66-fold increases in CPT production together with significantly high biomass yield. The study is helpful in the scale-up studies of O. mungos cell suspension culture in suitable bioreactor systems for the production of CPT.

  9. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract Background Embryonic stem cells (ESCs can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs. However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC

  10. Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Mahanom Jalil

    2015-01-01

    Full Text Available Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

  11. Functional Thyroid Follicular Cells Differentiation from Human-Induced Pluripotent Stem Cells in Suspension Culture

    Directory of Open Access Journals (Sweden)

    Ayumi Arauchi

    2017-05-01

    Full Text Available The replacement of regenerated thyroid follicular cells (TFCs is a promising therapeutic strategy for patients with hypothyroidism. Here, we have succeeded in inducing functional TFCs from human-induced pluripotent stem cells (iPSCs in scalable suspension culture. Differentiation of iPSCs with Activin A treatment produced Sox17- and FoxA2-expressing definitive endodermal cells that also expressed thyroid transcription factors Pax8 and Nkx2-1. Further treatment with thyroid-stimulating hormone (TSH induced TFCs expressing various types of thyroid proteins including TSH receptor, sodium–iodide symporter, thyroglobulin, and thyroid peroxidase. Interestingly, differentiated cells secreted free thyroxine in vitro. These results indicate successful differentiation of human iPSCs to functional TFCs that may enable us to fabricate thyroid tissues for regenerative medicine and disease models.

  12. Evaluation of limonoid production in suspension cell culture of Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Elisângela Fumagali Gerolino

    Full Text Available ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L. Osbeck, Rutaceae and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds, callus cultures (originating from hypocotyls, cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight. Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

  13. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    Tabata M, Mizukami H, Hiroaka N and Konoshima M 1974. Pigment formation in callus cultures of Lithospermum erythrorizon; Phytochemistry 13 927–932. Vander Esch S A, Giagnacovo G, Meccioni O and Vitali F 1993. Preliminary results on the production of azadirachtin by plant tissue culture of Azadirachta indica; G. Bot.

  14. Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures.

    Science.gov (United States)

    Bertolini, Alberto; Petrussa, Elisa; Patui, Sonia; Zancani, Marco; Peresson, Carlo; Casolo, Valentino; Vianello, Angelo; Braidot, Enrico

    2016-10-26

    Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

  15. Effects of auxins on growth and scopoletin accumulation in cell suspension cultures of Angelica archangelica L.

    Science.gov (United States)

    Siatka, T; Kasparová, M

    2008-01-01

    Scopoletin is a coumarin possessing many interesting biological effects, e.g., spasmolytic, anti-inflammatory, antimutagenic, antioxidant, antifungal, apoptosis-inducing, antiproliferative, acetylcholinesterase-inhibitory, and hypouricemic activities. Plant tissue cultures represent a promising alternative source of valuable plant-derived substances. A number of physical and chemical factors influence the cell growth and secondary metabolite biosynthesis in plant tissue cultures. The mechanism of their action is not completely understood. Besides other factors, plant growth regulators and light conditions play an important role. Effects of four auxins (2,4-dichlorophenoxyacetic acid, 2,4-D, alpha-naphthaleneacetic acid, NAA, beta-indoleacetic acid, IAA or beta-indolebutyric acid, IBA) at four concentrations (0.2, 2, 10 or 20 mg/l) on the culture growth and accumulation of scopoletin in the medium were tested in Angelica archangelica cell suspension cultures cultured under continuous light or in the dark. The highest culture growth was achieved with 2 mg/l 2,4-D, and 10 mg/l IAA. The best scopoletin levels were obtained with 0.2 mg/l 2,4-D, 2 mg/l 2,4-D, 10 mg/l NAA, and 20 mg/l IAA. The effects of light conditions were less marked than those of auxins and their concentrations in influencing both the cell growth and scopoletin accumulation in Angelica archangelica cell suspension cultures. The changes brought about by auxins were modified by light conditions.

  16. Somatic Embryogenesis of Date Palm (Phoenix dactylifera L.) Through Cell Suspension Culture.

    Science.gov (United States)

    Naik, Poornananda M; Al-Khayri, Jameel M

    2016-01-01

    Date palm (Phoenix dactylifera L.) is the oldest and most economically important plant species distributed in the hot arid regions of the world. Propagation of date palm by seeds produces heterogeneous offspring with inferior field performance and poor fruit quality. Traditionally, date palm is propagated by offshoots, but this method is inefficient for mass propagation because of limited availability of offshoots. Plant regeneration through tissue culture is able to provide technologies for the large-scale propagation of healthy true-to-type plants. The most commonly used technology approach is somatic embryogenesis which presents a great potential for the rapid propagation and genetic resource preservation of this species. Significant progress has been made in the development and optimization of this regeneration pathway through the establishment of embryogenic suspension cultures. This chapter focuses on the methods employed for the induction of callus from shoot tip explants, establishment of cell suspension culture, and subsequent somatic embryogenesis and plant regeneration.

  17. [Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor].

    Science.gov (United States)

    Li, Hui-hua; Yao, De-heng; Xu, Jian; Wang, Wei; Chang, Qiang; Su, Ming-hua

    2015-05-01

    Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.

  18. Isolation of fatty acids and aromatics from cell suspension cultures of Lavandula angustifolia.

    Science.gov (United States)

    Topçu, Gülaçti; Herrmann, Gabriele; Kolak, Ufuk; Gören, C; Porzel, Andrea; Kutchan, Toni M

    2007-02-01

    Cell suspension cultures of Lavandula angustifolia Mill. ssp. angustifolia (syn.: L. officinalis Chaix.) afforded a fatty acid composition, cis and trans p-coumaric acids (=p-hydroxy cinnamic acids), and beta-sitosterol. The fatty acid composition was analyzed by GC-MS, and the structures of the isolated three compounds were determined by 1H- and 13C-NMR, and MS spectroscopic techniques.

  19. Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Feria-Romero, Iris; Lazo, Elizabeth; Ponce-Noyola, Teresa; Cerda-García-Rojas, Carlos M; Ramos-Valdivia, Ana C

    2005-06-01

    Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

  20. Photosynthetic carbon metabolism in photoautotrophic cell suspension cultures grown at low and high CO2

    International Nuclear Information System (INIS)

    Roeske, C.A.; Widholm, J.M.; Ogren, W.L.

    1989-01-01

    Photosynthetic carbon metabolism was characterized in four photoautotrophic cell suspension cultures. There was no apparent difference between two soybeans (Glycine max) and one cotton (Gossypium hirsutum) cell line which required 5% CO 2 for growth, and a unique cotton cell line that grows at ambient CO 2 (660 microliters per liter). Photosynthetic characteristics in all four lines were more like C 3 mesophyll leaf cells than the cell suspension cultures previously studied. The pattern of 14 C-labeling reflected the high ratio of ribulosebisphosphate carboxylase to phosphoenolpyruvate carboxylase activity and showed that CO 2 fixation occurred primarily by the C 3 pathway. Photorespiration occurred at 330 microliters per liter CO 2 , 21% O 2 as indicated by the synthesis of high levels of 14 C-labeled glycine and serine in a pulse-chase experiment and by oxygen inhibition of CO 2 fixation. Short-term CO 2 fixation in the presence and absence of carbonic anhydrase showed CO 2 , not HCO 3 - , to be the main source of inorganic carbon taken up by the low CO 2 -requiring cotton cells. The cells did not have a CO 2 -concentrating mechanism as indicated by silicone oil centrifugation experiments. Carbonic anhydrase was absent in the low CO 2 -requiring cotton cells, present in the high CO 2 -requiring soybean cell lines, and absent in other high CO 2 cell lines examined. Thus, the presence of carbonic anhydrase is not an essential requirement for photoautotrophy in cell suspension cultures which grow at either high or low CO 2 concentrations

  1. Neuropharmacological and neuroprotective activities of some metabolites produced by cell suspension culture of Waltheria americana Linn.

    Science.gov (United States)

    Mundo, Jorge; Villeda-Hernández, Juana; Herrera-Ruiz, Maribel; Gutiérrez, María Del Carmen; Arellano-García, Jesús; León-Rivera, Ismael; Perea-Arango, Irene

    2017-10-01

    Waltheria americana is a plant used in Mexican traditional medicine to treat some nervous system disorders. The aims of the present study were to isolate and determine the neuropharmacological and neurprotective activities of metabolites produced by a cell suspension culture of Waltheria americana. Submerged cultivation of W. americana cells provided biomass. A methanol-soluble extract (WAsc) was obtained from biomass. WAsc was fractionated yielding the chromatographic fractions 4WAsc-H 2 O and WAsc-CH 2 Cl 2 . For the determination of anticonvulsant activity in vivo, seizures were induced in mice by pentylenetetrazol (PTZ). Neuropharmacological activities (release of gamma amino butyric acid (GABA) and neuroprotection) of chromatographic fractions were determined by in vitro histological analysis of brain sections of mice post mortem. Fraction 4WAsc-H 2 O (containing saccharides) did not produce neuronal damage, neurodegeneration, interstitial tissue edema, astrocytic activation, nor cell death. Pretreatment of animals with 4WAsc-H 2 O and WAsc-CH 2 Cl 2 from W. americana cell suspensions induced an increase in: GABA release, seizure latency, survival time, neuroprotection, and a decrease in the degree of severity of tonic/tonic-clonic convulsions, preventing PTZ-induced death of up to 100% of animals of study. Bioactive compounds produced in suspension cell culture of W. americana produce neuroprotective and neuropharmacological activities associated with the GABAergic neurotransmission system. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.

    Science.gov (United States)

    Singh, Harmeet; Mok, Pamela; Balakrishnan, Thavamalar; Rahmat, Siti Norfiza Binte; Zweigerdt, Robert

    2010-05-01

    We have systematically developed single cell-inoculated suspension cultures of human embryonic stem cells (hESC) in defined media. Cell survival was dependent on hESC re-aggregation. In the presence of the Rho kinase inhibitor Y-27632 (Ri) only approximately 44% of the seeded cells were rescued, but an optimized heat shock treatment combined with Ri significantly increased cell survival to approximately 60%. Mechanistically, our data suggest that E-cadherin plays a role in hESC aggregation and that dissociation and re-aggregation upon passaging functions as a purification step towards a pluripotency markers-enriched population. Mass expansion of hESC was readily achieved by up-scaling 2 ml cultures to serial passaging in 50 ml spinner flasks. A media comparison revealed that mTeSR was superior to KnockOut-SR in supporting cell proliferation and pluripotency. Persistent expression of pluripotency markers was achieved for two lines (hES2, hES3) that were used at higher passages (>86). In contrast, rapid down regulation of Oct4, Tra-1-60, and SSEA4 was observed for ESI049, a clinically compliant line, used at passages 20-36. The up-scaling strategy has significant potential to provide pluripotent cells on a clinical scale. Nevertheless, our data also highlights a significant line-to-line variability and the need for a critical assessment of novel methods with numerous relevant cell lines. Copyright 2010 Elsevier B.V. All rights reserved.

  3. Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

    Science.gov (United States)

    Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

    2017-02-01

    Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  4. Comparison of Cuminaldehyde Contents from Cell Suspension Cultures and Seeds of [Bunium persicum (Boiss. B. Fedtsch.

    Directory of Open Access Journals (Sweden)

    Sara KHOSRAVINIA

    2012-11-01

    Full Text Available The cell suspension culture and seed samples of Bunium persicum were extracted by supercritical fluid, hydrodistillation and solvent methods and analyzed by Gas Chromatography. In this study to compare the different methods of extractions, cuminaldehyde was targeted as one of the Black zira essential oil constitute. For callus induction the germinated seeds were cultured as explants on Murashige and Skoog medium supplemented with 2 mg/l 2,4-dichlorophenoxy acetic acid and 0.5 mg/l kinetin (treatment A as well as 2 mg/l ?-naphthalene acetic acid and 0.5 mg/l 6-benzyl aminopurine (treatment B and followed by cells suspension cultures establishment for the first time. The results of cell culture showed that cells from treatment B have a growth rate higher than A. All extracts were dissolved in 1 ml hexane and analyzed by Gas Chromatography. According to the Gas Chromatography analysis, cuminaldehyde was not detected in the supercritical fluid samples, while it was present in hydrodistillation and solvent extract. Cuminaldehyde percentage in cell and seed solvent extracts was 4.65% and 18.61% respectively. Gas Chromatography results also showed that no cuminaldehyde is present in media extracts, means no cuminaldehyde has been secreted into the medium.

  5. [Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims].

    Science.gov (United States)

    Cristovam, Priscila Cardoso; Glória, Maria Aparecida da; Melo, Gustavo Barreto; Gomes, José Alvaro Pereira

    2008-01-01

    To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

  6. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  7. Accumulation of phenylpropanoid derivatives in chitosan-induced cell suspension culture of Cocos nucifera.

    Science.gov (United States)

    Chakraborty, Moumita; Karun, Anitha; Mitra, Adinpunya

    2009-01-01

    Chitosan-induced elicitation responses of dark-incubated Cocos nucifera (coconut) endosperm cell suspension cultures led to the rapid formation of phenylpropanoid derivatives, which essentially mimics the defense-induced biochemical changes in coconut palm as observed under in vivo conditions. An enhanced accumulation of p-hydroxybenzoic acid as the major wall-bound phenolics was evident. This was followed by p-coumaric acid and ferulic acid. Along with enhanced peroxidases activities in elicited lines, the increase in activities of the early phenylpropanoid pathway enzymes such as, phenylalanine ammonia lyase (PAL), p-coumaroyl-CoA ligase (4CL) and p-hydroxybenzaldehyde dehydrogenase (HBD) in elicited cell cultures were also observed. Furthermore, supplementation of specific inhibitors of PAL, C4H and 4CL in elicited cell cultures led to suppressed accumulation of p-hydroxybenzoic acid, which opens up interesting questions regarding the probable route of the biosynthesis of this phenolic acid in C. nucifera.

  8. Production of Monascus pigments as extracellular crystals by cell suspension culture.

    Science.gov (United States)

    Lu, Fengling; Liu, Lujie; Huang, Yaolin; Zhang, Xuehong; Wang, Zhilong

    2018-01-01

    It is generally accepted that Monascus pigments are predominantly cell-bound, including both intracellular and surface-bound pigments. This long-term misconception was corrected in the present work. Production of extracellular crystal pigments by submerged culture of Monascus sp. was confirmed by microscopic observation and collection of Monascus pigments from extracellular broth by direct membrane filtration. Following up the new fact, the bioactivity of mycelia as whole-cell biocatalyst for biosynthesis and biodegradation of Monascus pigments had been detailedly examined in both an aqueous solution and a nonionic surfactant micelle aqueous solution. Based on those experimental results, cell suspension culture in an aqueous medium was developed as a novel strategy for accumulation of high concentration of Monascus pigments. Thus, glucose feeding during submerged culture in the aqueous medium was carried out successfully and high orange Monascus pigments concentration of near 4 g/L was achieved.

  9. pH and expansin action on single suspension-cultured tomato (Lycopersicon esculentum) cells.

    Science.gov (United States)

    Wang, C X; Wang, L; McQueen-Mason, S J; Pritchard, J; Thomas, C R

    2008-09-01

    The aim of this study was to measure key material properties of the cell walls of single suspension-cultured plant cells and relate these to cell-wall biochemistry. To this end, micromanipulation was used to compress single tomato cells between two flat surfaces until they ruptured, and force-deformation data were obtained. In addition to measuring the bursting force, we also determined the elastic (Young's) modulus of the cell walls by matching low strain (composites and proposed mechanisms of such failure. Through the measurement of cell-wall material properties using micromanipulation, it may be possible to understand more fully how cell-wall composition, structure and biochemistry lead to cell mechanical behaviour.

  10. Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

    Science.gov (United States)

    Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

    2012-12-20

    Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis - total protein content, thiols - reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

  11. Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Rene Kizek

    2012-12-01

    Full Text Available Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles, electronics (high-resolution imaging, logical circuits on the molecular level and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases or imaging (contrast agents. Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs and modified magnetic nanoparticles (MNPs on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis — total protein content, thiols — reduced (GSH and oxidized (GSSG glutathione, phytochelatins PC2-5, glutathione S-transferase (GST activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension

  12. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

  13. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

    Science.gov (United States)

    Thiruvengadam, Muthu; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Lee, Taek-Jun; Kim, Seung-Hyun; Chung, Ill-Min

    2016-01-01

    Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities. PMID:27854330

  14. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum

    Directory of Open Access Journals (Sweden)

    Muthu Thiruvengadam

    2016-11-01

    Full Text Available Anthraquinones (AQs and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs, media, sucrose, l-glutamine, jasmonic acid (JA, and salicylic acid (SA for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM; 3 and 2.93 g dry mass (DM and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds from cell suspension cultures, and the phytochemicals can be used for various biological activities.

  15. Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum.

    Science.gov (United States)

    Thiruvengadam, Muthu; Rekha, Kaliyaperumal; Rajakumar, Govindasamy; Lee, Taek-Jun; Kim, Seung-Hyun; Chung, Ill-Min

    2016-11-16

    Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum . We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum . The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum . This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities.

  16. A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

    Science.gov (United States)

    Kolewe, Martin E; Roberts, Susan C; Henson, Michael A

    2012-02-01

    The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance. Copyright © 2011 Wiley Periodicals, Inc.

  17. Production of triterpene acids by cell suspension cultures of Olea europaea.

    Science.gov (United States)

    Saimaru, Hiroshi; Orihara, Yutaka; Tansakul, Pimpimon; Kang, Young-Hwa; Shibuya, Masaaki; Ebizuka, Yutaka

    2007-05-01

    Olive (Olea europaea) contains large quantity of triterpene acids including oleanolic acid (6) as a major one. Varieties of biological activities exhibited by triterpene acids attracted our attentions, especially from pharmaceutical viewpoints. Cell culture of olive plant was induced and its triterpene constituents were studied. From the cell suspension cultures, six ursane type triterpene acids; ursolic acid (9), pomolic acid (10), rotundic acid (11), tormentic acid (12), 2alpha-hydroxyursolic acid (13) and 19alpha-hydroxyasiatic acid (14), and two oleanane type acids; oleanolic acid and maslinic acid (7), have been isolated. Quantity of ursane type triterpene acids produced by cell cultures was larger than that of oleanane type. Further, a multifunctional oxidosqualene cyclase (OSC) named OEA was cloned by homology based PCRs from the same cultured cells. Major product of OEA is alpha-amyrin (ursane skeleton), showing good accordance to higher content of ursane-type triterpene acids in the cultured cells, and strongly suggesting OEA to be a major contributor OSC for their production.

  18. Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Elke Gabriel

    Full Text Available Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in

  19. [The production of gastrodin through biotransformation of p-hydroxybenzaldehyde by cell suspension culture of Datura stramonium].

    Science.gov (United States)

    Gong, Jia-Shun; Ma, Wei-Peng; Pu, Jun-Xue; Xu, Shu-Guan; Zheng, Shuang-Qing; Xiao, Chun-Jie

    2006-10-01

    To investigate the production of p-hydroxymethylphenol-beta-D-glucoside (gastrodin) through biotransformation by plant cell suspension cultures. Using cell suspension cultures of Datura stramonium to convert the exogenous p-hydroxybenzaldehyde into gastrodin was conducted and the converted compounds were separated with a combination of multi-chromatography. Their chemical structures were determined on the basis of spectral analysis and chemical evidence. The conversion procedure of p-hydroxybenzaldehyde into gastrodin by Datura stramonium cell suspension cultures was established. The synthesized gastrodin (II) was isolated from the fermental liquor and identified by spectral analysis. At the same time, the p-hydroxybenzyl alcohol (I) converted through biotransformation of p-hydroxybenzaldehyde by cell suspension cultures of Datura stramonium was also isolated and identified. Two compounds were also isolated from the cell cultures and they were identified as beta-D-furanoallulose (III) and n-butyloxystyryl-beta-D-pyranoallulose (IV). Datura stramonium grown in suspension cultures can convert exogenous p-hydroxybenzaldehyde into the corresponding gastrodin.

  20. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Flores-Sánchez, Isvett J; Ortega-López, Jaime; del Carmen Montes-Horcasitas, María; Ramos-Valdivia, Ana C

    2002-12-01

    Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

  1. [Formation of protodioscin and deltoside isomers in suspension cultures of Nepal yam (Dioscorea deltoidea Wall.) cells].

    Science.gov (United States)

    Khandy, M T; Titova, M V; Konstantinova, S V; Kochkin, D V; Ivanov, I M; Nosov, A M

    2016-01-01

    Changes in the content of the furostanol glycosides protodioscin and deltoside, particularly that of the (25S)-isomers of the glycosides, during suspension cultivation of different lines of Nepal yam (Dioscorea deltoidea Wall.) cells of the strain IFR-DM-0.5 has been investigated. The composition of furostanol glycosides has been characterized, and the dynamics of the accumulation of individual glycosides during lengthy subcultivation of cells maintained in flasks or in a barbotage bioreactor has been analyzed. A positive correlation between the growth and accumulation of substances that belonged to the class of furostanol glycosides has been demonstrated for cultured dioscorea cells, whereas the content of some of the individual glycosides varied considerably between the lines of the strain, cultures maintained under different conditions, and even between cells in different phases of the growth cycle. The increased content of (25R)-forms of the glycosides (protodioscin and deltoside) was correlated with a decrease in the cellular growth rate, whereas an increase in culture growth intensity occurred concomitantly to an increase of the amount of (25S)-isomers. This may be indicative of the specific stimulatory effect of (25S)-glycosides, but not the (25R)-forms, on cell proliferation in vitro. Thus, the concentration of (25S)-forms may increase due to the autoselection of cells capable of intensive division during prolonged cultivation.

  2. Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : I. Induction and Purification.

    Science.gov (United States)

    Marques, I A; Brodelius, P E

    1988-09-01

    l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO(2) produced per minute per milligram protein at pH 8.4 and 30 degrees C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 +/- 300 daltons.

  3. Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures 1

    Science.gov (United States)

    Marques, Ivano A.; Brodelius, Peter E.

    1988-01-01

    l-Tyrosine decarboxylase (EC 4.1.1.25) activity was induced in cell suspension cultures of Thalictrum rugosum Ait. and Eschscholtzia californica Cham. with a yeast polysaccharide preparation (elicitor). The highest l-tyrosine decarboxylase activity in extracts from 7-day-old cell cultures of E. californica was observed 5 hours after addition of 30 to 40 micrograms elicitor per gram cell fresh weight. The enzyme extracted from cells of E. californica was purified 1540-fold to a specific activity of 2.6 micromoles CO2 produced per minute per milligram protein at pH 8.4 and 30°C. Purified enzyme from T. rugosum showed a specific activity of 0.18 micromoles per minute per milligram protein. The purification procedure involved ammonium sulfate fractionation, anion-exchange fast protein liquid chromatography, ultrafiltration, and hydrophobic interaction chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme from the two plant cell cultures had subunits of identical molecular weight (56,300 ± 300 daltons. Images Fig. 5 Fig. 6 PMID:16666277

  4. Desiccation-Enhanced Maturation and Germination of Date Palm Somatic Embryos Derived from Cell Suspension Culture.

    Science.gov (United States)

    Boufis, Nazim; Titouh, Khayreddine; Khelifi, Lakhdar

    2017-01-01

    In vitro plant regeneration via somatic embryogenesis is a powerful tool for rapid, large-scale production of healthy true-to-type plants. This approach is suitable to preserve existing natural genetic variability and propagation of variability generated from genetic improvement programs, including crossing, somaclonal variation, mutagenesis, and somatic hybridization. This chapter outlines a simplified protocol for date palm regeneration via somatic embryogenesis induced in cell suspension cultures. In this protocol, culture medium composition is manipulated, including plant growth regulators and solid (addition of agar) and liquid media to achieve reduction of production cycle of somatic embryogenesis, which increases the multiplication rate of embryogenic callus and improves the quantity and quality of somatic embryos.

  5. Inverse problem analysis of pluripotent stem cell aggregation dynamics in stirred-suspension cultures

    Science.gov (United States)

    Rostami, Mahboubeh Rahmati; Wu, Jincheng; Tzanakakis, Emmanuel S.

    2015-01-01

    The cultivation of stem cells as aggregates in scalable bioreactor cultures is an appealing modality for the large-scale manufacturing of stem cell products. Aggregation phenomena are central to such bioprocesses affecting the viability, proliferation and differentiation trajectory of stem cells but a quantitative framework is currently lacking. A population balance equation (PBE) model was used to describe the temporal evolution of the embryonic stem cell (ESC) cluster size distribution by considering collision-induced aggregation and cell proliferation in a stirred-suspension vessel. For ESC cultures at different agitation rates, the aggregation kernel representing the aggregation dynamics was successfully recovered as a solution of the inverse problem. The rate of change of the average aggregate size was greater at the intermediate rate tested suggesting a trade-off between increased collisions and agitation-induced shear. Results from forward simulation with obtained aggregation kernels were in agreement with transient aggregate size data from experiments. We conclude that the framework presented here can complement mechanistic studies offering insights into relevant stem cell clustering processes. More importantly from a process development standpoint, this strategy can be employed in the design and control of bioreactors for the generation of stem cell derivatives for drug screening, tissue engineering and regenerative medicine. PMID:26036699

  6. More for less: Improving the biomass yield of a pear cell suspension culture by design of experiments.

    Science.gov (United States)

    Rasche, Stefan; Herwartz, Denise; Schuster, Flora; Jablonka, Natalia; Weber, Andrea; Fischer, Rainer; Schillberg, Stefan

    2016-03-18

    Plant cell suspension cultures are widely used for the production of recombinant proteins and secondary metabolites. One of the most important steps during process development is the optimization of yields by testing different cultivation parameters, including the components of the growth medium. However, we have shown that the biomass yield of a cell suspension culture derived from the pear cultivar Pyrus communis cv. Champagner Bratbirne can be significantly improved solely by varying the temperature, inoculum density, illumination, and incubation time. In contrast to medium optimization, these simple physical factors are easily controlled and varied, thereby reducing the effort required. Using an experimental design approach, we improved the biomass yield from 146 g fresh weight (FW)/L to 407 g FW/L in only 5 weeks, simultaneously reducing the costs of goods sold per kg biomass from € 125 to € 45. Our simple approach therefore offers a rapid, efficient and economical process for the optimization of plant cell suspension cultures.

  7. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  8. Statistical experimental designs for the production of secondary metabolites in plant cell suspension cultures.

    Science.gov (United States)

    Schmitz, Christian; Fritsch, Leonie; Fischer, Rainer; Schillberg, Stefan; Rasche, Stefan

    2016-12-01

    Statistical experimental designs, also known as the "design of experiments" (DoE) approach, are widely used to improve not only technical processes but also to answer questions in the agricultural, medical and social sciences. Although many articles have been published about the application of DoE in these fields, few studies have addressed the use of DoE in the plant sciences, particularly in the context of plant cell suspension cultures (PCSCs). Compounds derived from PCSCs can be developed as pharmaceuticals, chemical feedstocks and cosmetic ingredients, and statistical experimental designs can be used to improve the productivity of the cells and the yield and/or quality of the target compounds in a cost efficient manner. In this article, we summarize recent findings concerning the application of statistical approaches to improve the performance of PCSCs and discuss the potential future applications of this approach.

  9. Metabolic labeling and membrane fractionation for comparative proteomic analysis of Arabidopsis thaliana suspension cell cultures.

    Science.gov (United States)

    Szymanski, Witold G; Kierszniowska, Sylwia; Schulze, Waltraud X

    2013-09-28

    Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% (1). Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient (2). Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K(15)NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest (3). By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or

  10. Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.

  11. Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.

    Science.gov (United States)

    Luna-Palencia, Gabriela R; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

  12. Effects of oligosaccharides from endophytic Fusarium oxysporum Dzf17 on activities of defense-related enzymes in Dioscorea zingiberensis suspension cell and seedling cultures

    Directory of Open Access Journals (Sweden)

    Peiqin Li

    2014-07-01

    Conclusions: Both EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.

  13. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...

  14. Induction of linalool as a pharmaceutical and medicinal metabolite via cell suspension culture of cumin (Cuminum cyminum L.).

    Science.gov (United States)

    Kazemi, N; Kahrizi, D; Mansouri, M; Karim, H; Vaziri, S; Zargooshi, J; Khanahmadi, M; Shokrinia, M; Mohammadi, N

    2016-05-30

    Cumin is an important medicinal plant in Iran. Plant cell suspension culture is a method for the production of medicinal and secondary metabolites. The linalool is a plant secondary metabolite that has been recognized as a neuroprotective agent. The purpose of this study was to evaluate the effects of salicylic acid elicitor on induction of linalool in cell suspension culture of cumin. For this purpose, the cumin seeds were prepared, to obtain sterile seedling, were disinfected with sodium hypochlorite and alcohol, and were cultured on MS basal medium. This research was conducted in two separate experiments including callus induction and suspension cultures. Leaf explants were prepared from sterile seedlings and used to produce callus on MS medium supplemented with 1 mg/l NAA and 0.5 mg/l BAP. In order to establish suspension culture, the appropriate calli were transferred to liquid medium. Then cell cultures were treated with elicitors. The effects of elicitor on the production of linalool secondary metabolite and cell viability were assessed by GC-Mass and tetrazolium test respectively. For this purpose, the salicylic acid (at concentrations of 0, 1, 2, 4 and 8 mg/l) was used. The experimental design was a completely randomized design with five treatments and three replications. The results of cell culture and GC-Mass analysis showed that salicylic acid had significant effects on the linalool production (linalool as a secondary metabolite and pharmaceutical agent in cell culture of cumin. It is necessary to determine the best combination of medium and elicitor.

  15. Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron

    International Nuclear Information System (INIS)

    Doolittle, R.L.; Richter, G.W.

    1981-01-01

    Cultures of Kupffer cells and of hepatocytes, prepared from single rat livers, synthesized ferritin protein equally efficiently. In culture but not in suspension, both sorts of cells responded significantly to stimulation with iron by increased ferritin synthesis. As determined by isoelectric focusing, the isoferritin profiles of newly synthesized 14 -labeled Kupffer cell and hepatocyte ferritin were identical, each having three bands. However, unlabeled ferritin, extracted from nonparenchymal liver cells (mainly Kupffer and endothelial cells) of iron-loaded rats, contained an acidic isoferritin that was not present in hepatocyte ferritin. Investigation of ferritin synthesis in cultured peritoneal and alveolar macrophages yielded similar results. The isofocusing profile of newly synthesized peritoneal macrophage ferritin was indistinguishable from the profile of fresh Kupffer cell or hepatocyte ferritin. Thus, the three isoferritins common to Kupffer cells, hepatocytes, and extrahepatic macrophages are neither cell- nor tissue-specific. However, modifications on intracellular storage may affect the isofocusing properties. The findings, although consistent with the LnH24-n subunit model of ferritin protein, indicate identical restrictive genomic control of the H:L ratios in these sorts of cells. Further, they make it probable that Kupffer cell ferritin iron, originating by endogenous synthesis, is the principal source of Kupffer cell hemosiderin iron

  16. Biosynthesis of camalexin from tryptophan pathway intermediates in cell-suspension cultures of Arabidopsis.

    Science.gov (United States)

    Zook, M

    1998-12-01

    Camalexin (3-thiazol-2'-yl-indole) is the principal phytoalexin that accumulates in Arabidopsis after infection by fungi or bacteria. Camalexin accumulation was detectable in Arabidopsis cell-suspension cultures 3 to 5 h after inoculation with Cochliobolus carbonum (Race 1), and then increased rapidly from 7 to 24 h after inoculation. Levels of radioactivity incorporated into camalexin during a 1.5-h pulse labeling with [14C]anthranilate also increased with time after fungal inoculation. The levels of radioactive incorporation into camalexin increased rapidly between 7 and 18 h after inoculation, and then decreased along with camalexin accumulation. Relatively low levels of radioactivity from [14C]anthranilate incorporated into camalexin in the noninoculated controls. Autoradiographic analysis of the accumulation of chloroform-extractable metabolites labeled with [14C]anthranilate revealed a transient increase in the incorporation of radioactivity into indole in fungus-inoculated Arabidopsis cell cultures. The time-course measurement of radioactive incorporation into camalexin during a 1.5-h pulse labeling with [14C]indole was similar to that with [14C]anthranilate. These data suggest that indole destined for camalexin synthesis is produced by a separate enzymatic reaction that does not involve tryptophan synthase.

  17. Biosynthesis of Camalexin from Tryptophan Pathway Intermediates in Cell-Suspension Cultures of Arabidopsis1

    Science.gov (United States)

    Zook, Michael

    1998-01-01

    Camalexin (3-thiazol-2′-yl-indole) is the principal phytoalexin that accumulates in Arabidopsis after infection by fungi or bacteria. Camalexin accumulation was detectable in Arabidopsis cell-suspension cultures 3 to 5 h after inoculation with Cochliobolus carbonum (Race 1), and then increased rapidly from 7 to 24 h after inoculation. Levels of radioactivity incorporated into camalexin during a 1.5-h pulse labeling with [14C]anthranilate also increased with time after fungal inoculation. The levels of radioactive incorporation into camalexin increased rapidly between 7 and 18 h after inoculation, and then decreased along with camalexin accumulation. Relatively low levels of radioactivity from [14C]anthranilate incorporated into camalexin in the noninoculated controls. Autoradiographic analysis of the accumulation of chloroform-extractable metabolites labeled with [14C]anthranilate revealed a transient increase in the incorporation of radioactivity into indole in fungus-inoculated Arabidopsis cell cultures. The time-course measurement of radioactive incorporation into camalexin during a 1.5-h pulse labeling with [14C]indole was similar to that with [14C]anthranilate. These data suggest that indole destined for camalexin synthesis is produced by a separate enzymatic reaction that does not involve tryptophan synthase. PMID:9847113

  18. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    Science.gov (United States)

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016. © 2016 American Institute of Chemical Engineers.

  19. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  20. Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco1

    Science.gov (United States)

    Fujita, Satoshi; Uchimura, Seiichi; Noguchi, Masahiro; Demura, Taku

    2016-01-01

    Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of α-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in α-tubulin can be used to prepare particular isotypes and specifically engineered versions of α-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures. PMID:26747285

  1. Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Dipto

    2012-05-01

    Full Text Available Abstract Background Podophyllotoxin (PTOX, the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA elicitation. High-resolution two-dimensional gel electrophoresis (2-DE followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell’s proteome. Result The HPLC analysis showed approximately 7–8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed. Conclusions Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.

  2. Efficient Secretion of Recombinant Proteins from Rice Suspension-Cultured Cells Modulated by the Choice of Signal Peptide

    Science.gov (United States)

    Huang, Li-Fen; Tan, Chia-Chun; Yeh, Ju-Fang; Liu, Hsin-Yi; Liu, Yu-Kuo; Ho, Shin-Lon; Lu, Chung-An

    2015-01-01

    Plant-based expression systems have emerged as a competitive platform in the large-scale production of recombinant proteins. By adding a signal peptide, αAmy3sp, the desired recombinant proteins can be secreted outside transgenic rice cells, making them easy to harvest. In this work, to improve the secretion efficiency of recombinant proteins in rice expression systems, various signal peptides including αAmy3sp, CIN1sp, and 33KDsp have been fused to the N-terminus of green fluorescent protein (GFP) and introduced into rice cells to explore the efficiency of secretion of foreign proteins. 33KDsp had better efficiency than αAmy3sp and CIN1sp for the secretion of GFP from calli and suspension-cultured cells. 33KDsp was further applied for the secretion of mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) from transgenic rice suspension-cultured cells; approximately 76%–92% of total rice-derived mGM-CSF (rmGM-CSF) was detected in the culture medium. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60. The extracellular yield of rmGM-CSF reached 31.7 mg/L. Our study indicates that 33KDsp is better at promoting the secretion of recombinant proteins in rice suspension-cultured cell systems than the commonly used αAmy3sp. PMID:26473722

  3. Plasma Membrane Proteomics of Arabidopsis Suspension-Cultured Cells Associated with Growth Phase Using Nano-LC-MS/MS.

    Science.gov (United States)

    Li, Bin; Takahashi, Daisuke; Kawamura, Yukio; Uemura, Matsuo

    2018-01-01

    Arabidopsis thaliana suspension-cultured cells (T87 line) are important model system for studies of responses to biotic and abiotic stresses at the cellular level in vitro since the cells have certain advantages compared with the whole plant system. However, the physiological and morphological characteristics of the cells are influenced by the progress of the growth phase of cells, which may result in different stress tolerance. To obtain comprehensive proteome profiles of the plasma membrane of Arabidopsis thaliana T87 suspension-cultured cells at the lag, log, or stationary growth phase, a shotgun proteomics method using nano-LC-MS/MS is used. The results obtained indicate that proteome profiles of the plasma membrane with the progress of the growth phase of cells dynamically changed, which may be associated with the physiological and morphological characteristics of the plasma membrane of the suspension-cultured cells. The proteomics results are further applied to explain different responsive patterns in the plasma membrane to cold acclimation and ABA treatment, which lead to understanding of different freezing tolerance associated with the growth phase of the cells.

  4. Effects of mercury (II) species on cell suspension cultures of catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, L. (Hangzhou Univ. (China)); Cullen, W.R. (Univ. of British Columbia, Vancouver, British Columbia (Canada))

    1994-11-01

    Mercury has received considerable attention because of its high toxicity. Widespread contamination with mercury poses severe environmental problems despite our extensive knowledge of its toxicity in living systems. It is generally accepted that the toxicity of mercury is related to its oxidation states and species, the organic forms being more toxic than the inorganic forms. In the aquatic environment, the toxicity of mercury depends on the aqueous speciation of the mercuric ion (Hg[sup 2+]). Because of the complex coordination chemistry of mercury in aqueous systems, the nature of the Hg[sup 2+] species present in aquatic environments is influenced greatly by water chemistry (e. g, pH, inorganic ion composition, and dissolved organics). Consequently, the influence of environmental factors on the aqueous speciation of mercury has been the focus of much attention. However, there is very little information available regarding the effects of the species and speciation on Hg (II) toxicity in plant-tissue cultures. Catharanthus roseus (C. roseus), commonly called the Madagascar Periwinkle, is a member of the alkaloid rich family Apocynaceae. The present investigation was concerned with the toxicity of mercury on the growth of C. roseus cell suspension cultures as influenced by mercury (II) species and speciation. The specific objectives of the study were to (a) study the effects of mercury species on the growth of C. roseus cultures from the point of view of environmental biology and toxicology; (b) evaluate the effects of selenate, selenite and selected ligands such as chloride, 1-cysteine in the media on the acute toxicity of mercuric oxide; (c) determine the impact of the initial pH of the culture media on the toxicities of mercuric compounds; (d) discuss the dependence of the toxicity on the chemical species and speciation of Hg (II). 11 refs., 7 figs., 2 tabs.

  5. Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells.

    Science.gov (United States)

    Nikolay, Alexander; Castilho, Leda R; Reichl, Udo; Genzel, Yvonne

    2017-03-23

    The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21 SUS ) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV PE ) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21 SUS cells with ZIKV PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×10 3 PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×10 7 cells/mL, and virus titers of 3.9×10 7 PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards

  6. Critical factors besides treatment dose and duration need to be controlled in Pb toxicity tests in plant cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Klaudia Sychta

    2017-08-01

    When effective illumination was adjusted to correct for turbidity at the highest lead concentration and pH was adjusted to 5.7–5.8, cell viability decreased with the increase of Pb(NO32 concentration and with treatment time. These experiments demonstrate that the toxic action of lead on cells in suspension depends strongly on culture conditions, and not only on the metal concentration and duration of treatment.

  7. Elicitor-Induced l-Tyrosine Decarboxylase from Plant Cell Suspension Cultures : II. Partial Characterization.

    Science.gov (United States)

    Marques, I A; Brodelius, P E

    1988-09-01

    Properties of purified l-tyrosine decarboxylase (EC 4.1.1.25) from elicitor-induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. l-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 +/- 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4 for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated l-tyrosine and l-beta-3,4-dihydroxyphenylalanine but was inactive toward l-phenylalanine and l-tryptophan. Apparent K(m) values of Eschscholtzia- and Thalictrum-decarboxylase for l-tyrosine were 0.25 +/- 0.03 and 0.27 +/- 0.04 millimolar, respectively. Similar affinities were found for l-3,4-dihydroxyphenylalanine. Eschscholtzial-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue l-alpha-aminooxy-beta-phenylpropionate and largely unaffected by d,l-alpha-monofluoromethyl-3,4-dihydroxyphenylalanine and alpha-difluoromethyltyrosine.

  8. Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Thu V. Vuong

    2014-06-01

    Full Text Available Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA, salicylic acid, β-glucan (GLU, and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

  9. Enhanced Production of Bioactive Isoprenoid Compounds from Cell Suspension Cultures of Artemisia annua L. Using β-Cyclodextrins

    Science.gov (United States)

    Rizzello, Francesca; De Paolis, Angelo; Durante, Miriana; Blando, Federica; Mita, Giovanni; Caretto, Sofia

    2014-01-01

    Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-β-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. PMID:25338048

  10. Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

    OpenAIRE

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2014-01-01

    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withan...

  11. Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

    Science.gov (United States)

    Lattanzio, Vincenzo; Caretto, Sofia; Linsalata, Vito; Colella, Giovanni; Mita, Giovanni

    2018-03-16

    Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  12. Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

    Science.gov (United States)

    Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

    2011-07-01

    The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

  13. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  14. Critical factors besides treatment dose and duration need to be controlled in Pb toxicity tests in plant cell suspension cultures

    OpenAIRE

    Klaudia Sychta; Janusz Szklarzewicz; Aneta Słomka; Ewa Gregoraszczuk; Elżbieta Kuta

    2017-01-01

    The study was designed to determine the proper conditions for suspension culture of Viola tricolor cells in toxicity studies of Pb at different concentrations (0, 200, 500, 1000, 2000 µM) and exposure times (24, 48, 72 h). By forming insoluble salts with ions from the medium, lead (II) nitrate added to the medium decreased the initial 5.7–5.8 pH of the medium, depending on the Pb salt concentration and light intensity. In alamarBlue assays, we found no dose- or time-dependent effect of Pb...

  15. Biotransformation of artemisinin using cell suspension cultures of Catharanthus roseus (L.) G.Don and Lavandula officinalis L.

    Science.gov (United States)

    Patel, Suman; Gaur, Rashmi; Verma, Priyanka; Bhakuni, Rajendra S; Mathur, Archana

    2010-08-01

    Artemisinin, an antimalarial compound, at 5 mg/40 ml, was transformed by cell suspension cultures of Catharanthus roseus (L.) G.Don and Lavandula officinalis L. into deoxyartemisinin with yields >78% (3.93 mg deoxyartemisinin from 5 mg artemisinin). Maximum conversion (78.6 and 78%) occurred after 6 and 7 days of adding artemisinin to 20 and 9 days old cultures of C. roseus and L. officinalis, respectively. The procedure was scaled up by and 500 mg artemisinin was transformed into 390 mg deoxyartemisinin. Addition of artemisinin at the beginning of the culture cycle resulted in >50% reduction in dry biomass production with no bioconversion. Conversion of artemisinin occurred intracellularly followed by leaching of the product into the medium.

  16. Role of polyamines in DNA synthesis of Catharanthus roseus cells grown in suspension culture

    Science.gov (United States)

    Rakesh Minocha; Subhash C. Minocha; Atsushi Komamine; Walter C. Shortle

    1990-01-01

    The requirement for polyamines in the proliferation of cells was first demonstrated in bacteria (3). While significant progress has been made in this field using animal cell cultures, only preliminary studies have been reported with plant tissues. Serafini-Fracassini et al. (9) showed a marked increase in polyamine synthesis early during the G 1 phase, concomitant with...

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

    Science.gov (United States)

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

    2016-08-07

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

  18. Elicitation of Jerusalem artichoke (Helianthus tuberosus L.) cell suspension culture for enhancement of inulin production and altered degree of polymerisation.

    Science.gov (United States)

    Ma, Chunquan; Zhou, Dong; Wang, Haitao; Han, Dongming; Wang, Yang; Yan, Xiufeng

    2017-01-01

    Plant cell suspension cultures have emerged as a potential source of secondary metabolites for food additives and pharmaceuticals. In this study inulin accumulation and its degree of polymerisation (DP) in the treated cells in the same medium were investigated after treatment with six types of elicitors. An in vitro cell suspension culture of Jerusalem artichoke (Helianthus tuberosus L.) was optimised by adding an extra nitrogen source. According to the growth kinetics, a maximum biomass of 5.48 g L -1 was obtained from the optimal cell suspension medium consisted of Murashige and Skoog basic medium (MS) + 1.0 mg L -1 α-naphthalene acetic acid (NAA) + 1.0 mg L -1 6-benzylaminopurine (6-BA) + 0.5 mg L -1 proline + 1.0 mg L -1 glutamine. Methyl jasmonate (MeJA, 250 µmol L -1 ) treatment for 15 days led to the highest levels of inulin (2955.27 ± 9.81 mg L -1 compared to control of 1217.46 ± 0.26 mg L -1 ). The elicited effect of five elicitors to the suspension cells of Jerusalem artichoke is as follows: AgNO 3 (Ag, 10 µmol L -1 ), salicylic acid (SA, 75 µmol L -1 ), chitosan (KJT, 40 mg L -1 ), Trichoderma viride (Tv, 90 mg L -1 ), yeast extract (YE, 0.25 mg L -1 ), and the corresponding content of inulin is increased by 2.05-, 1.93-, 1.76-, 1.44- and 1.18-fold compared to control, respectively. The obvious effect on the percentage of lower DP in inulin was observed in cells treated with 40 mg L -1 KJT, 0.25 mg L -1 YE and 10 µmol L -1 Ag. Among the six types of elicitors, the descending order of inulin content is MeJA > Ag > SA > KJT > Tv > YE. For the purpose inulin with lower DP and its application to prebiotic food, three elicitors, including KJT, YE and Ag, can be used for the elicitation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  19. Opposite extremes in ethylene/nitric oxide ratio induce cell death in suspension culture and root apices of tomato exposed to salt stress.

    Science.gov (United States)

    Poór, P; Borbély, P; Kovács, Judit; Papp, Anita; Szepesi, Ágnes; Takács, Z; Tari, Irma

    2014-12-01

    The plant hormone ethylene or the gaseous signalling molecule nitric oxide (NO) may enhance salt stress tolerance by maintaining ion homeostasis, first of all K+/Na+ ratio of tissues. Ethylene and NO accumulation increased in the root apices and suspension culture cells of tomato at sublethal salt stress caused by 100 mM NaCl, however, the induction phase of programmed cell death (PCD) was different at lethal salt concentration. The production of ethylene by root apices and the accumulation of NO in the cells of suspension culture did not increase during the initiation of PCD after 250 mM NaCl treatment. Moreover, cells in suspension culture accumulated higher amount of reactive oxygen species which, along with NO deficiency contributed to cell death induction. The absence of ethylene in the apical root segments and the absence of NO accumulation in the cell suspension resulted in similar ion disequilibrium, namely K+/Na+ ratio of 1.41 ± 0.1 and 1.68 ± 0.3 in intact plant tissues and suspension culture cells, respectively that was not tolerated by tomato.

  20. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L. Dunal in shake-flask culture and bioreactor.

    Directory of Open Access Journals (Sweden)

    Ganeshan Sivanandhan

    Full Text Available The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg, withanolide B (4826.05 mg, withaferin A (3732.81 mg, withanone (6538.65 mg, 12 deoxy withanstramonolide (3176.63 mg, withanoside IV (2623.21 mg and withanoside V (2861.18 mg] were achieved in the combined treatment of chitosan (100 mg/l and squalene (6 mM along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold as well as bioreactor (1.66-fold when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

  1. Enhanced Biosynthesis of Withanolides by Elicitation and Precursor Feeding in Cell Suspension Culture of Withania somnifera (L.) Dunal in Shake-Flask Culture and Bioreactor

    Science.gov (United States)

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2014-01-01

    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period. PMID:25089711

  2. Reactive oxygen and nitrogen (ROS and RNS) species generation and cell death in tomato suspension cultures--Botrytis cinerea interaction.

    Science.gov (United States)

    Pietrowska, E; Różalska, S; Kaźmierczak, A; Nawrocka, J; Małolepsza, U

    2015-01-01

    This article reports events connected to cell survival and Botrytis cinerea infection development in cell suspension cultures of two tomato cultivars which show different levels of susceptibility to the pathogen: cv. Corindo (more susceptible) and cv. Perkoz (less susceptible). In parallel changes in reactive oxygen (ROS) and nitrogen (RNS) species generation and in S-nitrosoglutathione reductase (GSNOR) activity were studied. In vivo staining methods with acridine orange (AO) and ethidium bromide (EB) as well as fluorescent microscopy were used to assess tomato and B. cinerea cells death. The biochemical studies of ROS and RNS concentrations in plant cell extract were complemented by in vivo ROS and nitric oxide (NO) imaging using nitro blue tetrazolium (NBT), diaminobenzidine (DAB) and diaminofluorescein diacetate (DAF-DA) staining methods, and confocal microscope technique. B. cinerea infection proceeded slower in Perkoz cell cultures. It was evidenced by measuring the pathogen conidia germination and germination tube development in which nuclei revealing cell death dominated. Two different types of tomato cell death were observed: cells with necrotic nuclei dominated in Corindo whereas in Perkoz cells with characteristic of vacuolar death type prevailed. In Perkoz cells, constitutive levels of NO and S-nitrosothiols (SNO) were significantly higher and hydrogen peroxide (H₂O₂) and superoxide anion (O₂(-)) concentrations were slightly higher as compared with Corindo cells. Moreover, increases in these molecule concentrations as a result of B. cinerea inoculation were observed in both, Perkoz and Corindo cell cultures. The enzymatic GSNOR activity seems to be an important player in controlling the SNO level in tomato cells. Involvements of the studied compounds in molecular mechanisms of tomato resistance to B. cinerea are discussed in the paper.

  3. Establishment of Cell Suspension Culture and Plant Regeneration in Abrus precatorius L., a Rare Medicinal Plant

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    Mohammad Serajur RAHMAN

    2012-02-01

    Full Text Available A new protocol has been developed for cell culture and in vitro regeneration of Abrus precatorius that holds enormous potentiality for preparation of medicines. In vitro grown calli were cultured in Murashige and Skoog (MS liquid media in agitated condition fortified with 0.5 mg/l 6-Benzylaminopurine. Growth curve of cells revealed that the cells continued to grow until 12 days of culture and got the highest peak from day 6-8. Isolated cell was found to produce highest 8.2% calli when suspended on MS medium supplemented with 0.5 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. Callus derived from single cell produced highest number of embryo (25-28% cultured on MS medium fortified with 2.0 mg/l 6-Benzylaminopurine and 0.2 mg/l 1-Naphthaleneacetic acid. The bipolar embryos were selected and optimum shoot formation was recorded on MS medium supplemented with 2.0 mg/l 6-Benzylaminopurine and 0.1 mg/l 1-Naphthaleneacetic acid. The optimum root induction was noticed in MS medium supplemented with 1.0 mg/l 3-Indolebutyric acid. Rooted plantlets were successfully transferred to potting soil and acclimatized to outdoor conditions.

  4. Accumulation of ixerin F and activities of some terpenoid bisynthetic enzymes in a cell suspension culture of Lactuca virosa L.

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    Anna Stojakowska

    2014-01-01

    Full Text Available A cell suspension culture of Lactuca virosa L. (Asteraceae, tribe Lactuceae is capable of synthesizing sesquiterpene lactones of which ixerin F is the main compound. The culture was characterized on growth (by dissimilation rates, on ixerin F accumulation (by RP-HPLC and on some enzyme activities involved in early steps of terpenoid biosynthesis. Acetoacetyl-coenzyme A thiolase (AACT, E.C. 2.3.1.9 and 3S-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS, E.C. 4.13.5 activities of the cells were assayed spectrophotometrically. HMGS activity increased during the culture period and reached a maximum during the stationary phase (190 pkat/mg protein, while AACT showed relatively high level of activity throughout the growth cycle, with transient decrease at the logarithmic growth phase and the beginning of stationary phase. Ixerin F accumulated inside the cells and the maximum concentration of 0.08% (on dry weight basis was found in the early stationary phase of the growth cycle of the culture.

  5. Effects of exogenous growth regulators on cell suspension culture of yin-hong grape (vitis vinifera l.) and establishment of the optimum medium

    International Nuclear Information System (INIS)

    Chao, Y.; Feng, J.C.; Yan, W.Y.; Xiao, Y.; Jun, Y.Y

    2015-01-01

    Callus induced by stem of Yin-hong grape (Vitis vinifera L.) was used as materials and B5 medium as basic medium. The major growth parameters of cell suspension cultures with various levels of 1-Naphthaleneacetic acid (NAA) and 6-Benzyl aminopurine (6-BA) were investigated to provide a basis for the optimum medium of suspension cell cultures of Yin-hong grape regarding cell number, packed cell volume (PCV), dry cell weight (DCW), cell viability, and morphology. All data were analysed by of two-way analysis of variance (ANOVA). Results showed that the treatment of 6-BA and NAA would effect the cell growth dynamics, probably causing logarithmic phase in advance at higher levels of 6-BA. Different concentration of 6-BA and NAA had significant effects on cells number, PCV, DCW and viability (p<0.05), while no-significant effect was observed on the cells morphology. The optimum medium for suspension cell cultures of Yin-hong grape was identified as B5+1.5 mg/L6-BA+1.5 mg/LNAA+ 250 mg/L casein hydrolysate + 30 g/L sucrose. With the optimum medium, the maximum number of suspension cells after the logarithmic growth phase was 34.78 * 108 / mL, the highest cell viability reached 86.45%.; DCW reached 3.84 g/L and PCV reached 0.092 mL/mL after eight days cultivating. (author)

  6. Characterization of an immunomodulatory Der p 2-FIP-fve fusion protein produced in transformed rice suspension cell culture.

    Science.gov (United States)

    Su, Chin-Fen; Kuo, I-Chun; Chen, Peng-Wen; Huang, Chiung-Hui; Seow, See Voon; Chua, Kaw Yan; Yu, Su-May

    2012-02-01

    Der p 2, a major allergen of Dermatophagoides pteronyssinus mites, is one of the most clinically relevant allergens to allergic patients worldwide. FIP-fve protein (Fve) from the golden needle mushroom (Flammulina velutipes) is an immunomodulatory protein with potential Th1-skewed adjuvant properties. Here, we produced and immunologically evaluated a Der p 2-Fve fusion protein as a potential immunotherapeutic for allergic diseases. Using an inducible expression system in cultured rice suspension cells, the recombinant Der p 2-Fve fusion protein (designated as OsDp2Fve) was expressed in rice cells under the control of an α-amylase gene (αAmy8) promoter and secreted under sucrose starvation. OsDp2Fve was partially purified from the cultured medium. The conformation of Der p 2 in OsDp2Fve remains intact as reflected by its unaltered allergenicity, as assessed by human IgE ELISA and histamine release assays, compared to non-fusion Der p 2 protein. Furthermore, the Fve protein expressed in OsDp2Fve retains its in vitro lymphoproliferative activity but loses its hemagglutination and lymphoagglutination effects compared to the native protein. Notably, in vivo evaluation showed that mice administered with OsDp2Fve possessed an enhanced production of Der p 2-specific IgG antibodies without potentiating the production of Der p 2-specific IgE and Th2 effector cytokines in comparison with mice co-administered with native Fve and Der p 2 proteins. These results suggest that the recombinant Der p 2-Fve fusion protein produced in rice suspension cell cultures has a great potential for allergy immunotherapy.

  7. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    Science.gov (United States)

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

  8. Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

    Science.gov (United States)

    Horiguchi, Ayumi; Yazaki, Koyuki; Aoyagi, Mami; Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

    2014-11-01

    Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Sepúlveda-Jiménez, Gabriela; Trejo-Espino, José Luis; Cerda-García-Rojas, Carlos M; de la Torre, Mayra; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2007-09-01

    Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

  10. The Effect of Plant Growth Regulators and Different Explants on the Response of Tissue Culture and Cell Suspension Cultures of German Chamomile (Matricaria chamomilla L.

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    L. Koohi,

    2014-07-01

    Full Text Available German chamomile (Matricaria chamomilla L. is one of the most important medicinal plants that its essential oils used in different medicinal industries. In this study which was carried out in 2013 growing season at the Faculty of Agricultural Sciences of the University of Mohaghegh Ardabili, the in vitro response of leaf and hypocotyl explants of German Chamomile in B5 medium supplemented with different levels of plant growth regulators including 2,4-D, naphthalene acetic acid (NAA, kinetin and 6-benzylaminopurine (BAP were investigated in a factorial experiment based on completely randomized design (CRD.In addition, cell suspension cultures were established and characterized. Hypocotyl and leaf explants exhibited cell proliferation and produced callus within 1-2 weeks. The highest fresh weight of the callus (264.1 mg was produced by leaf explants in the medium supplemented with 0.5 mg/l 2,4-D and 1 mg/l BAP. However, the leaf explants cultured on medium containing 1.5 mg/l 2,4-D showed the lowest cell proliferation and callus yield (40.42 mg. The highest percentage of root induction from leaf explants (58.73% was observed on the medium containing 4 mg/l 2,4-D and 1 mg/l Kin, and from hypocotyl explants (48.61% was observed on medium supplemented with 1.5 mg/l NAA. The 42.22% of calli derived from hypocotyl explants on B5 medium supplemented with 4 mg/l NAA and 3 mg/l BAP, were friable. Cell suspension cultures of German chamomile were established by transferring of hypocotyl-derived friable calli into the MS medium supplemented with 1.5 mg/l 2,4-D and 1 mg/l kinetin. The growth curve of cell proliferations started 4 days after culture and continued to grow until day 13th, where the cells entered stationary phase.

  11. Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

    Science.gov (United States)

    Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

    2017-10-01

    This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

  12. Role of Changes in Cell Fatty Acids Composition in the Increasing of Frost Resistance of Winter Wheat Suspension Culture

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    I.V. Lyubushkina

    2013-11-01

    Full Text Available Influences of low temperatures (4 and 8 ° С on the frost tolerance and fatty acid compositions of cells in a winter wheat suspension culture have been studied. It has been found that treatment of the culture with 4 °C (7 days did not protect cells from subsequent freezing temperature action (-8 °С, 6 h and was not accompanied significant changes in the fatty acid composition. On the contrary, the treatment of the culture with the temperature 8 °C (7 days prevented the death caused by freezing temperature and the content of saturated fatty acids decreased: pentadecanoic acid (by 35,0%, palmitic acid (by 19,9% and stearic acid (by 65,4%, and the content of α-linolenic acid increased by 94%. That was the cause of the double bond index (DBI increase by 16%. The role of fatty acids composition changes in the process of increasing frost tolerance in plants are discussed.

  13. Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch

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    Abinaya Manivannan

    2016-03-01

    Full Text Available Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS medium containing 3.0 mg·L−1 6-benzyladenine (BA in a combination with 2 mg·L−1 2,4-dichlorophenoxy acetic acid (2,4-D. From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa, salicylic acid (SA, and sodium nitroprusside (SNP on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

  14. Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch.

    Science.gov (United States)

    Manivannan, Abinaya; Soundararajan, Prabhakaran; Park, Yoo Gyeong; Jeong, Byoung Ryong

    2016-03-18

    Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS) medium containing 3.0 mg·L(-1) 6-benzyladenine (BA) in a combination with 2 mg·L(-1) 2,4-dichlorophenoxy acetic acid (2,4-D). From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa), salicylic acid (SA), and sodium nitroprusside (SNP) on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

  15. Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture.

    Science.gov (United States)

    Iranbakhsh, A R; Oshagi, M A; Ebadi, M

    2007-04-15

    Abstract: A number of physicochemical conditions such different concentration of glucose, sucrose, potassium nitrate, ammonium nitrate, calcium chloride and temperatures were tested to optimize growth and production of tropane alkaloids from Datura stramonium (Solanaceae) plants. Cell suspension from semi-clear calli of leave explants developed in MS medium containing kinetin (0.5 mg L(-1)) and NAA (2 mg L(-1)) hormones was used to measure biomass and total alkaloids and comparison of treatments. The results showed that 30 and 40 g L(-1) glucose led to the highest level of alkaloids and biomass productions, respectively. 20 and 40 g L(-1) sucrose concentrations resulted in order the most rates of alkaloids and biomass productions. The results showed that increasing of nitrate concentration led to the reduction of the alkaloids. The best concentration of potassium nitrate for the production of tropane alkaloids and biomass were in order 9.4 and 3.76 mM. Also it was evinced that the optimized concentration of ammonium nitrate for alkaloids production was 10.3 mM and for the biomass was 41.22 mM. The best concentration of calcium chloride for growth and production of the alkaloids was 7.92 mM. Testing different temperature specified that the best condition for production of the alkaloids was 20 degrees C whereas it was 25 degrees C for biomass production. The results of this study could be recommended to farmers involved in production of D. stramonium for tropain alkaloids at industrial and semi-industrial scales.

  16. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

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    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  17. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

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    Lenka Sangram K

    2012-04-01

    Full Text Available Abstract Background Taxol® (paclitaxel promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

  18. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

    Science.gov (United States)

    2012-01-01

    Background Taxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation. PMID:22530557

  19. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

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    Natalie Bordag

    Full Text Available The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli as well as mammalian cells chinese hamster ovary (CHO and mouse myeloma cells (NS0.The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  20. Enhanced production of vanillin flavour metabolites by precursor feeding in cell suspension cultures of Decalepis hamiltonii Wight & Arn., in shake flask culture.

    Science.gov (United States)

    Matam, Pradeep; Parvatam, Giridhar; Shetty, Nandini P

    2017-12-01

    The flavour rich tuberous roots of Decalepis hamiltonii are known for its edible and medicinal use and have become endangered due to commercial over-exploitation. Besides 2-Hydroxy-4-methoxy benzaldehyde (2H4MB), other flavour metabolites in tuberous roots include vanillin, 4-Methoxy Cinnamic acid derivatives, aromatic alcohols etc. So far, there are no reports on the pathway of 2H4MB biosynthesis nor there is an organized work on biotransformation using normal and cell suspension cultures for obtaining these metabolites using precursors. The main aim of the study is to develop a method for enhanced production of flavour attributing metabolites through ferulic acid (FA) feeding to the D. hamiltonii callus culture medium. Biomass of D. hamiltonii cell suspension cultures was maximum (200.38 ± 1.56 g/l) by 4th week. Maximum production of 2H4MB was recorded on 4th week (0.08 ± 0.01 mg/100 g dry weight) as quantified by HPLC. Addition of 0.1-1.5 mM ferulic acid as precursor in the culture medium showed significant ( p  vanillin, 2H4MB, vanillic acid, ferulic acid were of 0.1 ± 0.02 mg/100 g, 0.44 ± 0.01 mg/100 g, 0.52 ± 0.04 mg/100 g, 0.18 ± 0.02 mg/100 g DW respectively in 4 weeks of cultured cells supplemented with 1 mM ferulic acid as a precursor. The results indicate that, substantial increase in the levels of flavour metabolites in D. hamiltonii callus suspension culture was achieved. This would be having implications in biosynthesis of respective vanilla flavour attributing metabolites at very high levels for their large scale production.

  1. Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

    Science.gov (United States)

    Rakesh Minocha; Subhash C. Minocha; Stephanie L. Long; Walter C. Shortle

    1992-01-01

    Increased aluminum (Al) solubility in soil waters due to acid precipitation has aroused considerable interest in the problem of Al toxicity in plants. In the present study, an in vitro suspension culture system of Catharanthus roseus (L.) G. Don was used to analyze the effects of aluminum on several biochemical processes in these cells. The aliphatic...

  2. Changes in phytochelatins and their biosynthetic intermediates in red spruce (Picea rubens Sarg.) cell suspension cultures under cadmium and zinc stress

    Science.gov (United States)

    P. Thangavel; Stephanie Long; Rakesh Minocha

    2007-01-01

    Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC2 and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12...

  3. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Studies on suspension culture of virginia mallow

    Directory of Open Access Journals (Sweden)

    Anna Kasprzyk

    2014-04-01

    Full Text Available Virginia mallow (Sida hermaphrodita (L. Rusby belongs to the Malvaceae family. It is a very important industrial and energetic crop (Kasprzyk et al. 2013. In our studies, we used plant cell suspension cultures due to the fact that it is a useful tool to investigate biochemical, molecular and physiological aspects of many cellular functions (Dong et al. 2010. Virginia mallow seeds, obtained from Prof. Borkowska (University of Life Sciences in Lublin, Poland, were used in this investigation to obtain plants which were grown in sterile conditions in the Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University in Lublin, Poland. The seeds were surface sterilized and washed three times in sterile, distilled water. After 3 weeks of in vitro culture, young seedlings were used as a source of explants (to callus induction. Two types of explants were used to form callus culture: leaf and petiole. Callus tissues were then aseptically transferred to an Erlenmeyer flask with liquid medium and placed on an orbital shaker moving at 120 rpm. The observations of this suspense culture were conducted under light and confocal LSM microscopes. The authors observed that depending on the type of explants and composition of medium, callus tissue has varied in color and character of growth.

  5. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Directory of Open Access Journals (Sweden)

    Liping Ke

    Full Text Available Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel. In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L and bentazon (4.2 µmol. A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  6. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  7. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    International Nuclear Information System (INIS)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

    2014-01-01

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1 + -K cells) was established. Induction of Neurogenin3 expression in Nkx6.1 + -K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1 + -K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation

  8. Detection of anthraquinones and identification of 1,4-naphthohydroquinone in cell suspension cultures of Rudgea jasminoides (Cham.) Müll. Arg. (Rubiaceae)

    OpenAIRE

    Oliveira, Marisa de Cacia; Negri, Giusepina; Salatino, Antônio; Braga, Márcia Regina

    2007-01-01

    In Rubiaceae, anthraquinones and naphthoquinones are secondary metabolites characteristic of the subfamily Rubioideae, in which Rudgea jasminoides is included. Thin-layer chromatography using specific solvent systems and spray reagents indicated the presence of anthraquinones constitutively produced by cell suspension cultures of R. jasminoides. GC/MS analysis detected 1,4-naphthohydroquinone as a product of biosynthesis only after elicitation of the cells with yeast extract (Saccharomyces ce...

  9. Positive selection of Wharton's jelly-derived CD105(+) cells by MACS technique and their subsequent cultivation under suspension culture condition: A simple, versatile culturing method to enhance the multipotentiality of mesenchymal stem cells.

    Science.gov (United States)

    Amiri, Fatemeh; Halabian, Raheleh; Dehgan Harati, Mozhgan; Bahadori, Marzie; Mehdipour, Ahmad; Mohammadi Roushandeh, Amaneh; Habibi Roudkenar, Mehryar

    2015-05-01

    Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect alpha-smooth muscle actin (antigene) (α-SMA) protein. WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed α-SMA protein. In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.

  10. Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

    Science.gov (United States)

    Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

    2014-01-01

    Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

  11. Effects of zinc and cadmium ions on cell growth and production of coumarins in cell suspension cultures of Angelica archangelica L.

    Science.gov (United States)

    Siatka, Tomáš; Kašparová, Marie; Spilková, Jiřina

    2012-12-01

    The plant cell may respond to the excess of heavy metals in its environment by various mechanisms, including enhanced biosynthesis of secondary metabolites. In this study, zinc (0 to 1500 μM) and cadmium ions (0 to 100 μM) were tested as potential elicitors of the production of coumarins in angelica cell suspension cultures. In addition, the toxicity of both metals was assessed by evaluating their effect on cell growth (characterized by fresh and dry biomass at the end of a two-week subculture). It has been found that fresh biomass was not influenced up to zinc concentrations of 150 and 300 μM in the dark-grown and light-grown cultures, resp. Then it declined with an increasing zinc level. Zinc at 1500 μM diminished it by 54% and 24% in the dark-grown and light-grown cultures, resp. Dry biomass was influenced in a similar way. Zinc at 1500 μM reduced dry cell weight by 30% and 20% in cultures in the dark and in the light, resp. Cadmium ions did not affect fresh and dry weights of cells up to concentrations of 10 μM and 50 μM in cultures in the dark and in the light, resp. Toxic concentrations of cadmium are by an order of magnitude lower than those of zinc. Cadmium at 50 μM reduced fresh and dry cell weights by 66% and 59%, resp., in the dark-grown cultures. Cadmium at 100 μM caused a decrease in fresh and dry biomass by 40% and 44%, resp., in the light-grown cultures. Neither zinc nor cadmium improved production of coumarins.

  12. Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins

    Science.gov (United States)

    Conn, Simon; Curtin, Chris; Bézier, Annie; Franco, Chris; Zhang, Wei

    2008-01-01

    The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells. PMID:18836188

  13. Effects of 60Co γ-rays irradiation on cell growth and alkaloid accumulation of protocorm-like bodies in suspension cultures from Dendrobium huoshanense

    International Nuclear Information System (INIS)

    Hong Sali; Jin Qing; Huang Bei; Cai Yongping; Lin Yi

    2009-01-01

    Protocorm-like bodies (PLBs) in suspension cultures from Dendrobium huoshanense were irradiated by 60 Co γ-rays at doses of 5, 10, 20 and 30Gy, and alkaloid accumulation of PLBs was studied. The results showed that 60 Co γ-rays irradiation could improve the alkaloid content of PLBs, and the suitable dose was 10Gy. The fresh weight of 10Gy irradiated PLBs was 26.54g/flask, and the alkaloid content was 0.035% on the 36th day. The medium pH and electric conductivity of 10Gy irradiated PLBs changed slightly during the suspension culture period. The results suggested such cultural environment was suitable for PLBs growth continuely. Results also showed that 60 Co γ-rays irradiation could increase the activities of POD, SOD, CAT, PAL and decrease the activity of PPO, these were responsible for the improvement of cell growth and alkaloid accumulation in PLBs. (authors)

  14. A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization).

    Science.gov (United States)

    Baldwin, T. C.; McCann, M. C.; Roberts, K.

    1993-09-01

    Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.

  15. First insights in the Eu(III) speciation in plant cell suspension cultures

    International Nuclear Information System (INIS)

    Moll, Henry; Sachs, Susanne

    2017-01-01

    More than 80 % of the initial Eu(III) amount was associated on Brassica napus cells after an incubation time of 24 h. The spectroscopic speciation of the cell-bound Eu(III) is characterized by an increased intensity of the 7 F 2 transition and prolonged luminescence lifetimes.

  16. First insights in the Eu(III) speciation in plant cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Moll, Henry; Sachs, Susanne [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biogeochemistry

    2017-06-01

    More than 80 % of the initial Eu(III) amount was associated on Brassica napus cells after an incubation time of 24 h. The spectroscopic speciation of the cell-bound Eu(III) is characterized by an increased intensity of the {sup 7}F{sub 2} transition and prolonged luminescence lifetimes.

  17. Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (Festuca arundinacea) decreases cell wall feruloylation and increases rates of cell wall digestion.

    Science.gov (United States)

    Morris, Phillip; Dalton, Sue; Langdon, Tim; Hauck, Barbara; de Buanafina, Marcia M O

    2017-01-01

    In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase ( faeA ) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.

  18. Innovative, non-stirred bioreactors in scales from milliliters up to 1000 liters for suspension cultures of cells using disposable bags and containers--a Swiss contribution.

    Science.gov (United States)

    Werner, Sören; Eibl, Regine; Lettenbauer, Christine; Röll, Marcel; Eibl, Dieter; De Jesus, Maria; Zhang, Xiaowei; Stettler, Matthieu; Tissot, Stephanie; Bürki, Cedric; Broccard, Gilles; Kühner, Markus; Tanner, Rolf; Baldi, Lucia; Hacker, David; Wurm, Florian M

    2010-01-01

    Innovative mixing principles in bioreactors, for example using the rocking of a platform to induce a backwards and forwards 'wave', or using orbital shaking to generate a 'wave' that runs round in a cylindrical container, have proved to be successful for the suspension cultures of cells, especially when combined with disposable materials. This article presents an overview of the engineering characteristics when these new principles are applied in bioreactors, and case studies covering scales of operation from milliliters to 1000 liters.

  19. Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system

    Czech Academy of Sciences Publication Activity Database

    Maršík, Petr; Šíša, Miroslav; Lacina, O.; Moťková, Kateřina; Langhansová, Lenka; Rezek, Jan; Vaněk, Tomáš

    2017-01-01

    Roč. 220, JAN (2017), s. 383-392 ISSN 0269-7491 R&D Projects: GA ČR(CZ) GA14-22593S Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * Ibuprofen * Metabolism * Plant cells * Sequestration Subject RIV: CE - Biochemistry OBOR OECD: Plant sciences, botany Impact factor: 5.099, year: 2016

  20. Regeneration of soybean via embryogenic suspension culture

    Directory of Open Access Journals (Sweden)

    Droste Annette

    2001-01-01

    Full Text Available In an attempt to establish an alternative plant regeneration system for soybean [Glycine max (L. Merrill] cultivars used in Brazilian breeding programs, ten genotypes were tested for their embryogenic potential. Cotyledons were removed as explants from immature seeds harvested from field-grown plants. After 45 days on induction medium, the number of responding cotyledons and the number of somatic embryos per immature cotyledon were evaluated. The percentage of explants that produced somatic embryos varied from 1 to 70% among cultivars. The average number of somatic embryos produced per cotyledon pair ranged from 0.01 to 10.3 with a mean of 3.4. Suspension cultures were initiated with three Agrobacterium tumefaciens susceptible cultivars. Suspensions were successfully developed from Bragg and IAS5 cultivars. The packed cell volume, in one-month growth, increased 8.1 fold for Bragg and 3.5 fold for IAS5 and the fresh weight increased 6.6 and 2.8 fold, respectively. The cultivars differed for the analysed parameters. All tissue from each cultivar was transferred to the maturation medium and subsequently to the germination medium. The germination frequency was 45.7 and 54.9% for Bragg and IAS5, respectively. Plants were gradually exposed to ambient humidity over one week and then planted in soil. All plants yielded seeds in the greenhouse.

  1. Jasmonic Acid Effect on the Fatty Acid and Terpenoid Indole Alkaloid Accumulation in Cell Suspension Cultures of Catharanthus roseus

    Directory of Open Access Journals (Sweden)

    Guitele Dalia Goldhaber-Pasillas

    2014-07-01

    Full Text Available The stress response after jasmonic acid (JA treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA and terpenoid indole alkaloids (TIA. According to multivariate data analyses (MVDA, three major time events were observed and characterized according to the variations of specific FA and TIA: after 0–30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90–360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system.

  2. Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae

    Directory of Open Access Journals (Sweden)

    Koay Suan See

    2011-06-01

    Full Text Available Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of variousailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production.The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time oncell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of differentconcentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50mg/L had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45g/L sucrose without MeJA showed the highest pigment content (0.69±0.22Cv/g-FCM. The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40Cv/g-FCM. This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures. Rev. Biol. Trop. 59 (2: 597-606. Epub 2011 June 01.elastoma malabathricum pertenece a la familia de las melastomáceas, es una planta medicinal importante ampliamente distribuida desde Madagascar hasta Australia, que se utiliza en remedios tradicionales para el tratamiento de diversas dolencias. Además de sus propiedades medicinales, se ha identificado como una fuente potencial de producción de antocianinas. En esta

  3. Botulinum hemagglutinin-mediated in situ break-up of human induced pluripotent stem cell aggregates for high-density suspension culture.

    Science.gov (United States)

    Nath, Suman C; Tokura, Tomohiro; Kim, Mee-Hae; Kino-Oka, Masahiro

    2018-04-01

    Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high-density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break-up using botulinum hemagglutinin (HA), which specifically bound with E-cadherin and disrupted cell-cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E-cadherin-mediated cell-cell connections to facilitate the break-up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10 6 cells ml -1 was obtained by aggregate break-up into small ones, which was three times higher than that with the conventional culture without aggregate break-up. Therefore, the temporary activity of HA for disrupting E-cadherin-mediated cell-cell connection was key to establishing a simple in situ method for hiPSC aggregate break-up in bioreactors, leading to high cell density in suspension culture. © 2017 Wiley Periodicals, Inc.

  4. Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells.

    Science.gov (United States)

    Kimura, Yuta; Kuroda, Chie; Masuda, Kiyoshi

    2010-04-01

    NMCP1 is a plant protein that has a long coiled-coil domain within the molecule. Newly identified NMCP2 of Daucus carota and Apium graveolens showed similar peripheral localization in the interphase nucleus, and the sequence spanning the coiled-coil domain exhibited significant similarity with the corresponding region of NMCP1. To better understand disassembly and assembly of the nuclear envelope (NE) during mitosis, subcellular distribution of NMCP1 and NMCP2 was examined using A. graveolens cells. AgNMCP1 (NMCP1 in Apium) disassembled at prometaphase, dispersed mainly within the spindle, and accumulated on segregating chromosomes, while AgNMCP2 (NMCP2 in Apium), following disassembly at prometaphase with timing similar to that of AgNMCP1, dispersed throughout the mitotic cytoplasm at metaphase and anaphase. The protein accumulated at the periphery of reforming nuclei at telophase. A probe for the endomembrane indicated that the nuclear membrane (NM) disappears at prometaphase and begins to reappear at early telophase. Growth of the NM continued after mitosis was completed. NMCP2 in the mitotic cytoplasm localized in vesicular structures that could be distinguished from the bulk endomembrane system. These results suggest that NMCP1 and NMCP2 are recruited for NE assembly in different pathways in mitosis and that NMCP2 associates with NM-derived vesicles in the mitotic cytoplasm.

  5. Use of embryogenic cell suspension and meristem-tip cultures for mutation breeding of apomictic Musa species

    International Nuclear Information System (INIS)

    Novak, F.J.; Afza, R.; Duren, M. van

    1990-01-01

    Full text: Breeding by crossing is difficult for banana and plantain. The plants are heterozygous, therefore mutagenic treatment may uncover a recessive allele by mutating or deleting a corresponding dominant allele. Meristem tips were excised from in vitro growing shoots and used for mutation experiments. Induction was carried out by irradiating shoot tips with γ rays and/or by treatment of explants with ethylmethanesulfonate (EMS). Cell suspension was initiated from corm and leaf tissue excised from in vitro grown plantlets. Mutagenised cell suspensions were derived from leaf and corm tissues irradiated with 60 Co γ rays - (10 to 60 Gy, 8 Gy/min). Musa clones exhibited differences in radiosensitivity and post-irradiation recovery. Doses of 20 to 40 Gy seem suitable for mutation induction. The EMS concentration of 25 mM for 4 hours was found effective for isolated shoot tips. Considerable phenotypic variation was observed among plants regenerated from in vitro shoot tips after mutagenic treatment. Leaf and corm explants kept their morphogenic ability in embryogenic cell suspensions after irradiation up to 25 Gy. (author)

  6. Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model.

    Science.gov (United States)

    Gould, A R

    1977-01-01

    The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G2 and mitosis. G1 phase has a much lower Q10 than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263-1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.

  7. Effects of chilling on protein synthesis in tomato suspension cultures

    International Nuclear Information System (INIS)

    Matadial, B.; Pauls, K.P.

    1989-01-01

    The effect of chilling on cell growth, cell viability, protein content and protein composition in suspension cultures of L. esculentum and L. hirsutum was investigated. Cell growth for both species was arrested at 2 degrees C but when cultures were transferred to 25 degree C cell growth resumed. There was no difference in viability between control and chilled cultures of L. esculentum, however, L. hirsutum control cultures exhibited larger amounts of Fluorescein Diacetate induced fluorescence than chilled cultures. 35 S-methionine incorporation into proteins was 2.5-2 times higher in L. hirsutum than in L. esculentum. Quantitative and qualitative differences, in 35 S-methionine labelled proteins, between chilled and control cultures were observed by SDS-PAGE and fluorography. Protein content in chilled cultures decreased over time but then increased when cultures were transferred to 25 degrees C

  8. Cell Suspension Culture of Eriobotrya japonica Regulates the Diabetic and Hyperlipidemic Signs of High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Jin-Bin Wu

    2013-03-01

    Full Text Available The present study investigates the anti-hyperlipidemic and antihyperglycemic effects and mechanism in high-fat (HF-fed mice of cell suspension culture of Eriobotrya japonica (TA, which contains a great number of pentacyclic terpenoids. Firstly, C57BL/6J mice were randomly divided into two groups: the control (CON group was fed with a low-fat diet (n = 9, whereas the experimental group was fed a 45% HF diet for 8 weeks. Afterwards, the CON group was treated with vehicle, whereas the HF group was subdivided into five groups and was orally given TA or rosiglitazone or not for 4 weeks. Blood and visceral adipose tissue, liver tissue and skeletal muscle were examined. Treatment with TA reduced body weight gain, weights of white adipose tissue (WAT (including epididymal, perirenal, mesenteric WAT and visceral fat, and hepatic triacylglycerol content significantly without affecting food intake in diet-induced diabetic mice. TA effectively prevented HF diet-induced increases in the levels of blood glucose, insulin, leptin and HOMA-IR index (p < 0.001, p < 0.05, p < 0.05, p < 0.01, respectively and attenuated insulin resistance. Treatment with TA, adipocytes in the visceral depots showed a reduction in size. TA effectively significantly increased the protein contents of phosphorylation of AMPK-α (Thr172 both in liver and adipose tissue. It is shown that TA exhibits hypolipidemic effect in HF-fed mice by decreasing gene expressions of fatty acid synthesis, including acyl-coenzyme A: diacylglycerol acyltransferase (DGAT 2, which catalyzes the final step in the synthesis of triglycerides, and antidiabetic properties occurred as a result of decreased hepatic glucose production via phosphenolpyruvate carboxykinase (PEPCK down- regulation, improved insulin sensitization and TA (at 1.0 g/kg dose decreased expression of hepatic and adipose 11-β-hydroxysteroid dehydroxygenase (11β-HSD1 gene, which contributed in attenuating diabetic state. Futhermore, TA at

  9. Autologous epidermal cell suspension: A promising treatment for chronic wounds.

    Science.gov (United States)

    Zhao, Hongliang; Chen, Yan; Zhang, Cuiping; Fu, Xiaobing

    2016-02-01

    Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Skin grafting is an important treatment method for chronic wounds, which are refractory to conservative therapy. The technique involving epidermal cell suspensions was invented to enable the possibility of treating larger wounds with only a small piece of donor skin. Both uncultured and cultured autologous epidermal cell suspensions can be prepared and survive permanently on the wound bed. A systematic search was conducted of EMBASE, Cochrane Library, PubMed and web of science by using Boolean search terms, from the establishment of the database until May 31, 2014. The bibliographies of all retrieved articles in English were searched. The search terms were: (epithelial cell suspension OR keratinocyte suspension) and chronic and wound. From the included, 6 studies are descriptive interventions and discussed the use of autologous keratinocyte suspension to treat 61 patients' chronic wound. The various methods of preparation of epidermal cell suspension are described. The advantages and shortcomings of different carriers for epidermal cell suspensions are also summarised. Both uncultured and cultured autologous epidermal cell suspensions have been used to treat chronic wounds. Although the limitations of these studies include the small number of patient populations with chronic wounds and many important problems that remain to be solved, autologous epidermal cell suspension is a promising treatment for chronic wounds. Copyright © 2015 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  10. Induced accumulation of 20-hydroxyecdysone in cell suspension ...

    African Journals Online (AJOL)

    This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata, an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly ...

  11. Induced accumulation of 20-hydroxyecdysone in cell suspension ...

    African Journals Online (AJOL)

    Administrator

    2011-09-12

    Sep 12, 2011 ... This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata, an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production.

  12. Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts.

    Science.gov (United States)

    Emmerling, M; Seitz, H U

    1990-09-01

    A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc4Xyl3GalFuc). This nonasaccharide showed excellent "anti-auxin" properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 μM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.

  13. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. (Univ. of Illinois, Urbana (USA))

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I{sub 50} values for growth, chlorophyll (Chl), {beta}-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in ({sup 14}C)clomazone uptake cannot account for selectivity since there were significantly greater levels of domazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  14. The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

    International Nuclear Information System (INIS)

    Kwiatkowska, Aleksandra; Zebrowski, Jacek; Oklejewicz, Bernadetta; Czarnik, Justyna; Halibart-Puzio, Joanna; Wnuk, Maciej

    2014-01-01

    Highlights: • A decrease in proliferation rate during long-term cultivation of Arabidopsis cells. • Age-dependent increase in senescence-associated gene expression in Arabidopsis cells. • Age-related increase in DNA methylation, H3K9me2, and H3K27me3 in Arabidopsis cells. • High potential of photosynthetic efficiency of long-term cultured Arabidopsis cells. - Abstract: Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage

  15. Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor.

    Science.gov (United States)

    Ahlawat, Seema; Saxena, Parul; Ali, Athar; Khan, Shazia; Abdin, Malik Z

    2017-05-01

    Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in shake flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in shake flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in shake flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to shake flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Suspension culture process of MethA tumor cell for the production of heat-shock protein glycoprotein 96: process optimization in spinner flasks.

    Science.gov (United States)

    Tang, Ya-Jie; Li, Hong-Mei; Hamel, Jean-François P

    2007-01-01

    Heat-shock proteins (HSPs) act like "chaperones", making sure that the cell's proteins are in the right shape and in the right place at the right time. Heat-shock protein glycoprotein 96 (gp96) is a member of the HSP90 protein family, which chaperones a number of molecules in protein folding and transportation. Heat-shock protein gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Currently, heat-shock protein gp96 was only isolated from murine and human tissues and cell lines. An animal cell suspension culture process for the production of heat-shock protein gp96 by MethA tumor cell was developed for the first time in spinner flasks. Effects of culture medium and condition were studied to enhance the MethA tumor cell density and the production and productivity of heat-shock protein gp96. Initial glucose concentration had a significant effect on the heat-shock protein gp96 accumulation, and an initial glucose level of 7.0 g/L was desirable for MethA tumor cell growth and heat-shock protein gp96 production and productivity. Cultures at an initial glutamine concentration of 3 and 6 mM were nutritionally limited by glutamine. At an initial glutamine concentration of 6 mM, the maximal viable cell density of 19.90 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 4.95 mg/L was obtained. The initial concentration of RPMI 1640 and serum greatly affected the MethA tumor cell culture process. Specifically cultures with lower initial concentration of RPMI 1640 resulted in lower viable cell density and lower heat-shock protein gp96 production. At an initial serum concentration of 8%, the maximal viable cell density of 19.18 x 10(5) cells/mL and the maximal heat-shock protein gp96 production of 5.67 mg/L was obtained. The spin rate significantly affected the cell culture process in spinner flasks, and a spin rate of 150 rpm was desirable for MethA tumor cell growth and heat-shock protein gp96

  17. LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design.

    Science.gov (United States)

    Sarvin, Boris; Fedorova, Elizaveta; Shpigun, Oleg; Titova, Maria; Nikitin, Mikhail; Kochkin, Dmitry; Rodin, Igor; Stavrianidi, Andrey

    2018-03-30

    In this paper, the ultrasound assisted extraction method for isolation of steroidal glycosides from D. deltoidea plant cell suspension culture with a subsequent HPLC-MS determination was developed. After the organic solvent was selected via a two-factor experiment the optimization via Latin Square 4 × 4 experimental design was carried out for the following parameters: extraction time, organic solvent concentration in extraction solution and the ratio of solvent to sample. It was also shown that the ultrasound assisted extraction method is not suitable for isolation of steroidal glycosides from the D. deltoidea plant material. The results were double-checked using the multiple successive extraction method and refluxing extraction. Optimal conditions for the extraction of steroidal glycosides by the ultrasound assisted extraction method were: extraction time, 60 min; acetonitrile (water) concentration in extraction solution, 50%; the ratio of solvent to sample, 400 mL/g. Also, the developed method was tested on D. deltoidea cell suspension cultures of different terms and conditions of cultivation. The completeness of the extraction was confirmed using the multiple successive extraction method. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

    Science.gov (United States)

    Tapia, Felipe; Vogel, Thomas; Genzel, Yvonne; Behrendt, Ilona; Hirschel, Mark; Gangemi, J David; Reichl, Udo

    2014-02-12

    Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Effect of light wavelength on cell growth, content of phenolic compounds and antioxidant activity in cell suspension cultures of Thevetia peruviana.

    Science.gov (United States)

    Arias, J P; Zapata, K; Rojano, B; Arias, M

    2016-10-01

    Thevetia peruviana (T. peruviana) has been considered as a potentially important plant for industrial and pharmacological application. Among the number of compounds which are produced by T. peruviana, antioxidants and polyphenols are of particular interest due to their benefits on human health. Cell suspension cultures of T. peruviana were established under different conditions: 1) constant illumination (24h/day) at different light wavelengths (red, green, blue, yellow and white), 2) darkness and 3) control (12h/12h: day light/dark) to investigate their biomass, substrate uptake, polyphenols production and oxidizing activity. The results showed biomass concentrations between 17.1g dry weight (DW)/l (green light) and 18.2g DW/l (control) after 13days. The cultures that grew under green light conditions consumed completely all substrates after 10days, while other cultures required at least 13days or more. The total phenolic content was between 7.21 and 9.46mg gallic acid (GA)/g DW for all light conditions. In addition the ferric reducing antioxidant power and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid antioxidant activity ranged from 5.41-6.58mg ascorbic acid (AA)/g DW and 82.93-110.39μmol Trolox/g DW, respectively. Interestingly, the samples which grew under the darkness presented a higher phenolic content and antioxidant capacity when compared to the light conditions. All together, these results demonstrate the extraordinary effect of different lighting conditions on polyphenols production and antioxidant compounds by T. peruviana. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Physiological responses of suspension cultures of Catharanthus roseus to aluminum: changes in polyamines and inorganic ions

    Science.gov (United States)

    Xinhua Zhou; Rakesh Minocha; Subhash C. Minocha

    1995-01-01

    The effects of aluminum (Al) treatment on polyamines were studied using suspension cultures of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. The addition of A1 (0.2, 0.5, 1.0 mM) to the suspension cultures caused a significant increase in putrescine within 24h only in freshly transferred cells. By contrast, Al treatment reduced putrescine...

  1. Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids

    International Nuclear Information System (INIS)

    Kapur, S K; Wang, X; Shang, H; Yun, S; Li, X; Feng, G; Khurgel, M; Katz, A J

    2012-01-01

    Adipose-derived stromal/stem cells (ASCs) have been gaining recognition as an extremely versatile cell source for tissue engineering. The usefulness of ASCs in biofabrication is further enhanced by our demonstration of the unique properties of these cells when they are cultured as three-dimensional cellular aggregates or spheroids. As described herein, three-dimensional formulations, or self-assembling ASC spheroids develop their own extracellular matrix that serves to increase the robustness of the cells to mechanical stresses. The composition of the extracellular matrix can be altered based on the external environment of the spheroids and these constructs can be grown in a reproducible manner and to a consistent size. The spheroid formulation helps preserve the viability and developmental plasticity of ASCs even under defined, serum-free media conditions. For the first time, we show that multiple generations of adherent ASCs produced from these spheroids retain their ability to differentiate into multiple cell/tissue types. These demonstrated properties support the idea that culture-expanded ASCs are an excellent candidate cellular material for ‘organ printing’—the approach of developing complex tissue structures from a standardized cell ‘ink’ or cell formulation. (paper)

  2. Mevastatin-induced inhibition of cell growth in avocado suspension ...

    African Journals Online (AJOL)

    Cell suspension cultures were established using soft, friable callus derived from nucellar tissue of 'Hass' avocado (Persea americana Mill.) seed from fruit harvested 190 days after full bloom. Cell cultures were maintained in liquid medium supplemented with naphthalene acetic acid (NAA), isopentenyl adenine (iP) and ...

  3. Effect of NaCl on ionic content and distribution in suspension-cultured cells of the halophyte Sonneratia alba versus the glycophyte Oryza sativa.

    Science.gov (United States)

    Hayatsu, Manabu; Suzuki, Suechika; Hasegawa, Ai; Tsuchiya, Shinpei; Sasamoto, Hamako

    2014-09-15

    The effect of a high concentration of NaCl on the intra- (cytoplasmic matrix and vacuole) and extracellular (cell wall) distribution of Na, Cl, K, Mg, Ca, S, and P was investigated in suspension-cultured cells of the mangrove halophyte Sonneratia alba and compared to cultured cells of glycophytic rice (Oryza sativa). No significant differences were observed in ultrastructural features of cluster cells of both species cultured with and without 50mM NaCl. Quantitative X-ray microanalysis of cryosections of the cells cultured in the presence of 50mM NaCl showed that the Na concentration ([Na]) and Cl concentration ([Cl]) significantly increased in all three cell components measured. In S. alba, the [Na] was highest in the vacuole and lowest in the cytoplasmic matrix, while the [Cl] was highest in the cell wall and lowest in the cytoplasmic matrix. In O. sativa, however, the [Na] and [Cl] were highest in the cell wall, and the [Na] was lowest in the cytoplasmic matrix. Thus, the possible activities for Na and Cl transport from the cytoplasmic matrix into the vacuole were greater in S. alba than in O. sativa, suggesting that halophilic mangrove cells gain salt tolerance by transporting Na and Cl into their vacuoles. In O. sativa, the addition of NaCl to the culture medium caused no significant changes to the intracellular concentrations of various elements, such as K, P, S, Ca, and Mg, which suggests the absence of a direct relationship with the transport Na and Cl. In contrast, a marked decrease in the Ca concentration ([Ca]) in the cytoplasmic matrix and vacuole and an approximately two-fold increase in the P concentration ([P]) in the cytoplasmic matrix were found in S. alba, suggesting that the decrease in the [Ca] is related to the halophilic nature of S. alba (as indicated by the inward movement of Na(+) and Cl(-)). The possible roles of a Na(+)/Ca(2+) exchange mechanism in halophilism and the effect of the [P] on the metabolic activity under saline conditions are

  4. Enhancement of 20-hydroxyecdysone production in cell suspension ...

    African Journals Online (AJOL)

    ... most effective. The maximum 20-hydroxyecdysone productivity of about 1.31 mg/L/day was observed in culture with 10 mg/L 7-dehydrocholesterol. This data is the first indication that 7-dehydrocholesterol and ergosterol feeding are effective precursors for 20-hydroxyecdysone formation in plant cell suspension culture.

  5. Rice suspension cultured cells are evaluated as a model system to study salt responsive networks in plants using a combined proteomic and metabolomic profiling approach.

    Science.gov (United States)

    Liu, Dawei; Ford, Kristina L; Roessner, Ute; Natera, Siria; Cassin, Andrew M; Patterson, John H; Bacic, Antony

    2013-06-01

    Salinity is one of the major abiotic stresses affecting plant productivity but surprisingly, a thorough understanding of the salt-responsive networks responsible for sustaining growth and maintaining crop yield remains a significant challenge. Rice suspension culture cells (SCCs), a single cell type, were evaluated as a model system as they provide a ready source of a homogenous cell type and avoid the complications of multicellular tissue types in planta. A combination of growth performance, and transcriptional analyses using known salt-induced genes was performed on control and 100 mM NaCl cultured cells to validate the biological system. Protein profiling was conducted using both DIGE- and iTRAQ-based proteomics approaches. In total, 106 proteins were identified in DIGE experiments and 521 proteins in iTRAQ experiments with 58 proteins common to both approaches. Metabolomic analysis provided insights into both developmental changes and salt-induced changes of rice SCCs at the metabolite level; 134 known metabolites were identified, including 30 amines and amides, 40 organic acids, 40 sugars, sugar acids and sugar alcohols, 21 fatty acids and sterols, and 3 miscellaneous compounds. Our results from proteomic and metabolomic studies indicate that the salt-responsive networks of rice SCCs are extremely complex and share some similarities with thee cellular responses observed in planta. For instance, carbohydrate and energy metabolism pathways, redox signaling pathways, auxin/indole-3-acetic acid pathways and biosynthesis pathways for osmoprotectants are all salt responsive in SCCs enabling cells to maintain cellular function under stress condition. These data are discussed in the context of our understanding of in planta salt-responses. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

    Science.gov (United States)

    Islam, Md Reyazul; Kim, Nan-Sun; Jung, Jae-Wan; Kim, Hyo-Boon; Han, So-Chon; Yang, Moon-Sik

    2018-01-01

    A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH 2 -terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Mapping and characterisation of the sorghum cell suspension ...

    African Journals Online (AJOL)

    Here we reported the first secretomic study of sorghum (Sorghum bicolor), a naturally drought tolerant cereal crop. In this study, we used a gel-based proteomic approach in combination with mass spectrometry to separate and identify proteins secreted into the culture medium of sorghum cell suspensions, a first step ...

  8. Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Pedreño, María A

    2014-04-01

    Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  9. Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure.

    Science.gov (United States)

    Shin, Yun-Ji; Chong, Yun-Jo; Yang, Moon-Sik; Kwon, Tae-Ho

    2011-12-01

    The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  10. Nano-hydroxyapatite colloid suspension coated on chemically modified porous silicon by cathodic bias: a suitable surface for cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez, Alejandra [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Electroquimica y Energia Quimica de la Universidad de Costa Rica (CELEQ), Universidad de Costa Rica, 2060 (Costa Rica); Gonzalez, Jerson [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Garcia-Pineres, Alfonso [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Investigacion en Biologia Celular y Molecular (CIBCM), Universidad de Costa Rica, 2060 (Costa Rica); Montero, Mavis L. [Escuela de Quimica, Universidad de Costa Rica, 2060 (Costa Rica); Centro de Electroquimica y Energia Quimica de la Universidad de Costa Rica (CELEQ), Universidad de Costa Rica, 2060 (Costa Rica); Centro de Ciencia e Ingenieria en Materiales (CICIMA), Universidad de Costa Rica, 2060 (Costa Rica)

    2011-06-15

    The properties of porous silicon make it an interesting material for biological applications. However, porous silicon is not an appropriate surface for cell growth. Surface modification is an alternative that could afford a bioactive material. In this work, we report a method to yield materials by modification of the porous silicon surface with hydroxyapatite of nanometric dimensions, produced using an electrochemical process and coated on macroporous silicon substrates by cathodic bias. The chemical nature of the calcium phosphate deposited on the substrates after the experimental process and the amount of cell growth on these surfaces were characterized. (copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  11. Changes in gene expression during programmed cell death in tomato cell suspensions

    NARCIS (Netherlands)

    Hoeberichts, F.A.; Orzaez, D.; Plas, van der L.H.W.; Woltering, E.J.

    2001-01-01

    To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was

  12. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. 1

    Science.gov (United States)

    Funk, Christoph; Brodelius, Peter E.

    1990-01-01

    Feeding of cinnamic acid and ferulic acid to non-treated and chitosan-treated cell suspension cultures of Vanilla planifolia resulted in the formation of trace amounts of p-hydroxy benzoic acid (5.2 micrograms per gram fresh weight of cells) and vanillic acid (6.4 micrograms per gram fresh weight of cells), respectively. Addition of a 4-hydroxycinnamate: CoA-ligase inhibitor, 3,4-(methylenedioxy)-cinnamic acid (MDCA), resulted in a reduced biosynthesis of ligneous material with a simultaneous significant increased vanillic acid formation (around 75 micrograms per gram fresh weight of cells). A K1 of 100 micromolar for 4-hydroxycinnamate: CoA-ligase in a crude preparation was estimated for this inhibitor. It is suggested that the conversion of cinnamic acids into benzoic acids does not involve cinnamoyl CoA esters as intermediates. Feeding of 14C-cinnamic acid and 14C-ferulic acid to cells treated with MDCA indicate that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of V. planifolia. PMID:16667725

  13. Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

    Science.gov (United States)

    Caron, Angelo Luis; Biaggio, Rafael Tagé; Swiech, Kamilla

    2018-01-01

    Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

  14. Determination of cardiac glycosides and total phenols in different generations of Securigera securidaca suspension culture

    Directory of Open Access Journals (Sweden)

    Z. Tofighi

    2016-04-01

    Full Text Available Background and objectives: The seeds of Securigera securidaca (L. Deg. & Dorf. (Fabaceae are used as anti-diabetic remedy in Iranian folk medicine. The aim of the present study was to establish the callus and suspension culture of S. securidaca seeds for the first time and to determine the major secondary metabolites including cardiac glycosides and total phenols. Methods: The culture of S. securidaca from seeds was initiated in hormone-supplemented MS medium containing 1 and 0.1 ppm 2, 4-D solution for solid and suspension cultures, respectively, sucrose and vitamins (B1, B2, B6, Folic acid, Biotin, Nicotinamide and Ca pantothenate at 25 °C and 12 h photoperiods. The cardiac glycosides were determined based on the calibration curve of securidaside which was isolated from the seeds extract of S. securidaca. Total phenolic compounds of different generations of suspension culture were determined using Folin Ciocalteu reagent. Results: Callus culture of S. securidaca was grown light cream to pale yellow in color and soft in texture while the cells of suspension culture grew cream to yellow with isolated cells and small aggregates. The production of cardiac glycosides in the 7th generation were more than the seeds extract (p

  15. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

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

    2010-01-01

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

  16. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Yakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.J.; Harren, F.J.M.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 23 days which indicates the existence

  17. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.; Harren, F.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2¿3 days which indicates the existence

  18. Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

    OpenAIRE

    Yoshihiro, Ozeki; Atsushi, Komamine; Department of Botany, Faculty of Science, The University of Tokyo:(Present)Department of Biology, College of Arts and Sciences, The University of Tokyo; Department of Botany, Faculty of Science, The University of Tokyo:(Present)Biological Institute, Faculty of Science, Tohoku University

    1986-01-01

    The effects of plant growth regulators were investigated on anthocyanin synthesis induced by removing auxin from carrot suspension cultures. Of the auxins tested, 2,4-D showed the strongest inhibiting effect on anthocyanin synthesis and had the strongest promoting effect on undifferentiated growth. When 2,4-D was added to anthocyanin synthesizing cells, in which cell division had ceased, anthocyanin synthesis was repressed immediately, accumulated anthocyanin disappeared and cell division res...

  19. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. 1

    Science.gov (United States)

    Funk, Christoph; Brodelius, Peter E.

    1990-01-01

    Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-14C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia. PMID:16667674

  20. Purification and characterization of three chitinases and one beta-1,3-glucanase accumulating in the medium of cell suspension cultures of barley (Hordeum vulgare L.)

    DEFF Research Database (Denmark)

    Kragh, K.M.; Jacobsen, S.; Dalgaard Mikkelsen, J.

    1991-01-01

    -terminal sequence (24 residues) this enzyme was identified as the previously described beta-1,3-endoglucanase GII from germinating barley grain. The results suggest that secretion of chitinase and beta-1,3-glucanase from the embryogenic cell suspensions of barley corresponds to accumulation of the same enzymes...... chromatography. Two of the chitinases were identified as the previously described endochitinases T and C from barley grain. The third and novel chitinase, designated K, was the major basic chitinase in the medium constituting 4% of the soluble protein. Chitinase K was found to be a 33-kDa endochitinase with p......I at 8.7. Further analysis showed that this enzyme is also expressed in barley grain. The amino acid composition and five partial amino acid sequences covering 93 residues of chitinase K were determined. A high similarity was found between chitinase K and barley chitinase T and C as well as basic...

  1. Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions

    Science.gov (United States)

    Durzan, D. J.

    1999-01-01

    Haploid and diploid cell suspensions of Taxus spp. were examined for their adaptive plasticity in response to simulated microgravity, unit gravity, and hypergravity. Cell suspensions produced the taxane, paclitaxel, (TAXOL (R)), which is useful for the treatment of various cancers. Amyloplasts contributed to taxane ring biosynthesis and to drug release at the cell wall. Drug-producing cells reacted as gravisensing osmotic tensiometers. In stressed cells, amyloplasts docked and fused in clusters to sites on the plasmalemma before taxane discharge into the culture medium. In simulated microgravity and compared to all other treatments, taxane production was reduced nearly 100-fold. The percent paclitaxel of total taxanes remained 3-to 6-fold greater, and biomass doubled. When p53-independent programmed cell death was induced, taxanes were released into the culture medium as free molecules (soluble and insoluble) or bound to membranes, nuclear fragments, xylan residues, and other particulate materials. Unit gravity and especially hypergravity promoted xylogenesis and significant drug overproduction. A model relating families of >touch = (TCH), taxane early response (TER), nuclear cycling, and apoptosis-regulating genes to gravisensing, cell wall modifications, and to taxane recovery accounted for most but not all of the observations.

  2. The influence of glutamine on growth and viability of cell suspension cultures of Douglas-fir after exposure to polyethylene glycol.

    Science.gov (United States)

    Leustek, T; Kirby, E G

    1988-12-01

    The response of cell cultures of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) to osmotic stress was studied by measuring cell growth and viability after exposure to polyethylene glycol (PEG) (M(r) 6000-8000). Growth of cells inoculated in a medium containing 10% PEG was slightly inhibited, whereas growth in a medium containing 15% PEG was severely inhibited. Cells grown for 6 days in nutrient medium and then subcultured in a medium containing 15% PEG to induce water stress showed high viabilities, whereas cells grown for longer than 6 days before exposure to PEG showed decreased viabilities after subculture. Cells grown in medium containing 30 mM glutamine were significantly more resistant to PEG-induced water stress, as measured by viability, than cells grown in medium without glutamine.

  3. Establishment of embryogenic suspension cultures of Pinus radiata ...

    African Journals Online (AJOL)

    The development of embryonal suspensor mass (ESM) from immature embryos of Pinus radiata on a solidified growth medium containing 0, 5 mgl -1 benzyladenine, 3, 0 mgl -1 2, 4-dichlorophenoxyacetic acid, 500 mgl -1 casein hydrolysate and 250 mgl -1 L-glutamine was used as inoculum to establish cell suspension ...

  4. 2,3,5,4′- Tetrahydroxystilbene-2-O-β-D-glycoside Biosynthesis by Suspension Cells Cultures of Polygonum multiflorum Thunb and Production Enhancement by Methyl Jasmonate and Salicylic Acid

    Directory of Open Access Journals (Sweden)

    Li Shao

    2012-02-01

    Full Text Available Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA and kinetin (KT. Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L and 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glycoside (THSG, 56.39 mg/L of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA and salicylic acid (SA could increase THSG production. The most appropriate concentration of MeJA was 100 μmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L. The most appropriate concentration of SA was 125 μmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L.

  5. 2,3,5,4'- tetrahydroxystilbene-2-O-β-D-glycoside biosynthesis by suspension cells cultures of Polygonum multiflorum Thunb and production enhancement by methyl jasmonate and salicylic acid.

    Science.gov (United States)

    Shao, Li; Zhao, Shu-Jin; Cui, Tang-Bing; Liu, Zhong-Yu; Zhao, Wei

    2012-02-22

    Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA) and kinetin (KT). Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L) and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glycoside (THSG, 56.39 mg/L) of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA) and salicylic acid (SA) could increase THSG production. The most appropriate concentration of MeJA was 100 μmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L). The most appropriate concentration of SA was 125 μmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L).

  6. Suspension state increases reattachment of breast cancer cells by up-regulating lamin A/C.

    Science.gov (United States)

    Zhang, Xiaomei; Lv, Yonggang

    2017-12-01

    Extravasation is a rate-limiting step of tumor metastasis, for which adhesion to endothelium of circulating tumor cells (CTCs) is the prerequisite. The suspension state of CTCs undergoing detachment from primary tumor is a persistent biomechanical cue, which potentially regulates the biophysical characteristics and cellular behaviors of tumor cells. In this study, breast tumor cells MDA-MB-231 in suspension culture condition were used to investigate the effect of suspension state on reattachment of CTCs. Our study demonstrated that suspension state significantly increased the adhesion ability of breast tumor cells. In addition, suspension state markedly promoted the formation of stress fibers and focal adhesions and reduced the motility in reattached breast cancer cells. Moreover, lamin A/C was reversibly accumulated at posttranscriptional level under suspension state, improving the cell stiffness of reattached breast cancer cells. Disruption of actin cytoskeleton by cytochalasin D caused lamin A/C accumulation. Conversely, decreasing actomyosin contraction by ROCK inhibitor Y27632 reduced lamin A/C level. Knocking down lamin A/C weakened the suspension-induced increase of adhesion, and also abolished the suspension-induced decrease of motility and increase of stress fibers and focal adhesion in reattaching tumor cells, suggesting a crucial role of lamin A/C. In conclusion, it was demonstrated that suspension state promoted the reattachment of breast tumor cells by up-regulating lamin A/C via cytoskeleton disruption. These findings highlight the important role of suspension state for tumor cells in tumor metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. A rational approach for the improvement of biomass production and lipid profile in cacao cell suspensions.

    Science.gov (United States)

    Rúa, Adriana María Gallego; Rojas, Luisa Fernanda; Trujillo, Aura Ines Urrea; Zuleta, Oriana Parra; Alvarez, Cristian David Correa; Garcés, Lucía Atehortúa

    2017-06-23

    Cocoa butter (CB) is produced in the seeds of Theobroma cacao representing 50% of its dry weight. The lipid composition plays an important role in the physicochemical, rheological, and sensory properties of the CB, making this fat a valuable resource for the production of chocolates, cosmetics, and pharmaceuticals. In this paper, are described experimental strategies used for a rational improvement of biomass production and fatty acids in cacao cell suspension cultures. First, the lipid profile in four cacao varieties is characterized, and then, one variety is selected to induce cell suspensions using a direct method without previous establishment of a callus phase. To improve growth and total fat production in cell suspension cultures, modified DKW media and newly designed media culture, based on the mineral concentrations of cacao seeds (cacao biomass production, "CBP"), are analyzed and compared. In addition, the effect of acetate in the lipid profile of cell suspensions is evaluated. Ultrastructural histological analysis of lipid vesicles in cacao seeds and cell suspensions is also performed. The results will show that it is feasible to establish cacao suspensions without the calli step and increase the biomass production by selecting a suitable cacao variety and tissue and also applying a new culture media formulation. In addition, it is possible to synthesize fatty acids in cell cultures and modify the lipid profile adding a precursor of the novo biosynthesis of fatty acids such as the acetate. Transmission electronic microscopy examinations and differential interference contrast microscopy analysis will demonstrate that lipid vesicles are the main reserve substance in both cacao seeds and cell suspensions.

  8. Identification of Alternaria alternata Mycotoxins by LC-SPE-NMR and Their Cytotoxic Effects to Soybean (Glycine max Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Edson Rodrigues-Filho

    2013-02-01

    Full Text Available This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1, alternariol (2, 3'-hydroxyalternariol monomethyl ether (3, and alternariol monomethyl ether (4, were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max cell cultures as a model. EC50 values which range from 0.11 (±0.02 to 4.69 (±0.47 μM showed the high cytotoxicity of these compounds.

  9. Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

    International Nuclear Information System (INIS)

    Dong, C.-Z.; Montillet, J.-L.; Triantaphylides, C.

    1994-01-01

    The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells (Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H + -ATPase activity. Plasma membrane-enriched vesicles were prepared and the H + -ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H + -ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2mM dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H + -ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH. This rapid irreversible inhibition of the plasma membrane H + -ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material. (author)

  10. Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris Enzimas do metabolismo da sacarose em cultura celular de Bauhinia forficata, Curcuma zedoaria e Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Marcia Ometto de Mello

    2001-09-01

    Full Text Available The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.O objetivo deste trabalho foi estudar as enzimas do metabolismo da sacarose em culturas de célula em suspensão de Bauhinia forficata Link, Curcuma zedoaria Roscoe e Phaseolus vulgaris L. A via da invertase foi identificada nas três espécies estudadas. A via da sacarose sintase também foi responsável pelo metabolismo da sacarose em células de Curcuma zedoaria e Phaseolus vulgaris. Foram encontradas atividades maiores que 300 nmol min-1 mg-1 de proteína das enzimas invertase ácida e alcalina, UDPglicose pirofosforilase e fosfoglicomutase no extrato celular das três espécies de plantas. A sacarose sintase mostrou atividade baixa nas células de Bauhinia forficata. À medida que a concentração de sacarose no meio de cultura diminuiu, a atividade da sacarose sintase aumentou em células de Curcuma zedoaria e Phaseolus vulgaris. Ao final do período de cultura, quando os carboidratos se tornaram limitantes, as atividades das enzimas glicolíticas reduziram-se gradualmente.

  11. Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray.

    Science.gov (United States)

    Werry, P A; Stoffelsen, K M

    1978-07-01

    Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 μm. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.

  12. Passaging protocols for mammalian neural stem cells in suspension bioreactors.

    Science.gov (United States)

    Sen, Arindom; Kallos, Michael S; Behie, Leo A

    2002-01-01

    Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.

  13. Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

    DEFF Research Database (Denmark)

    Garcia-Angulo, P.; Willats, W. G. T.; Encina, A. E.

    2006-01-01

    showed an overall composition similar to that of non-habituated cells, with exception of an increase in glucose in hemicellulosic fractions tightly bound to cellulose. However, these cells also showed reduced levels of extensin and AGP labelling. These differences could be related to the high tolerance......The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension-cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence...

  14. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  15. Acoustic manipulation of bacteria cells suspensions

    Science.gov (United States)

    GutiéRrez-Ramos, Salomé; Hoyos, Mauricio; Aider, Jean Luc; Ruiz, Carlos; Acoustofluidics team Team; Soft; Bio group Collaboration

    An acoustic contacless manipulation gives advantages in the exploration of the complex dynamics enviroment that active matter exhibits. Our works reports the control confinement and dispersion of Escherichia coliRP437-pZA3R-YFP suspensions (M9Glu-Ca) via acoustic levitation.The manipulation of the bacteria bath in a parallel plate resonator is achieved using the acoustic radiation force and the secondary radiation force. The primary radiation force generates levitation of the bacteria cells at the nodal plane of the ultrasonic standing wave generated inside the resonator. On the other side, secondary forces leads to the consolidation of stable aggregates. All the experiments were performed in the acoustic trap described, where we excite the emission plate with a continuous sinusoidal signal at a frequency in the order of MHz and a quartz slide as the reflector plate. In a typical experiment we observed that, before the input of the signal, the bacteria cells exhibit their typical run and tumble behavior and after the sound is turned on all of them displace towards the nodal plane, and instantaneously the aggregation begins in this region. CNRS French National Space Studies, CONACYT Mexico.

  16. Acid-Fast Staining and Petroff Hausser Chamber Counting of Mycobacterial Cells in Liquid Suspension

    OpenAIRE

    Treuer, Robin; Haydel, Shelley E.

    2011-01-01

    Accurate and rapid cell counts of mycobacterial species in culture are difficult to obtain. Here, a method using modified Kinyoun acid-fast staining was adapted for use with a Petroff-Hausser sperm and bacteria cell counting chamber by using a liquid suspension staining technique. Cell counts obtained by this method were compared to viable cell counts by agar plate counting, revealing accurate correlation.

  17. In vitro morphogenesis and cell suspension culture establishment in Piper solmsianum C. DC. (Piperaceae Morfogênese in vitro e estabelecimento de culturas de suspensão celular em Piper solmsianum C. DC. (Piperaceae

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena

    2009-03-01

    Full Text Available Piper solmsianum is a shrub from Southeast Brazil in which many biologically active compounds were identified. The aim of this work was to establish a cell suspension culture system for this species. With this in mind, petiole and leaf explants obtained from in vitro plantlets were cultured in the presence of different plant growth regulator combinations (IAA, NAA, 2,4-D and BA. Root and indirect shoot adventitious formation, detected by histological analysis, was observed. Besides the different combinations of plant growth regulators, light regime and the supplement of activated charcoal (1.5 mg.l-1 were tested for callus induction and growth. Cultures maintained in light, on a 0.2 mg.l-1 2,4-D and 2 mg.l-1 BA supplemented medium, and in the absence of activated charcoal, showed the highest calli fresh matter increment. From a callus culture, cell suspension cultures were established and their growth and metabolite accumulation studied. The achieved results may be useful for further characterization of the activated secondary metabolites pathways in in vitro systems of P. solmsianum.Piper solmsianum é uma espécie herbácea do sudeste brasileiro onde vários compostos biologicamente ativos já foram identificados. O objetivo deste trabalho foi estabelecer suspensões celulares nesta espécie. Para tanto, foram utilizados explantes de pecíolos e folhas, retirados de plântulas cultivadas in vitro, os quais foram submetidos a diferentes combinações de reguladores de crescimento (AIA, ANA, 2,4-D e BAP. Foi obtida a neo-formação de raízes e brotos, estes últimos através do processo de organogênese indireta evidenciada por estudos histológicos. Para a indução e crescimento dos calos, foram avaliados, além das diferentes combinações de reguladores de crescimento, a suplementação ao meio de cultura de carvão ativado (1,5 mg.l-1 e o regime de luz. Culturas mantidas na luz, em meio de cultura suplementado com 0,2 mg.l-1 2,4-D e 2 mg

  18. Elicitor-induced tyrosine decarboxylase in berberine-synthesizing suspension cultures of Thalictrum rugosum.

    Science.gov (United States)

    Gügler, K; Funk, C; Brodelius, P

    1988-01-04

    Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.

  19. Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture

    Science.gov (United States)

    Johanson, Kelly; Allen, Patricia L.; Lewis, Fawn; Cubano, Luis A.; Hyman, Linda E.; Hammond, Timothy G.

    2002-01-01

    This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.

  20. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Directory of Open Access Journals (Sweden)

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  1. Effects of aluminum on growth, polyamine metabolism, and inorganic ions in suspension cultures of red spruce (Picea rubens)

    Science.gov (United States)

    Rakesh Minocha; Walter C. Shortle; Daniel J. Jr. Coughin; Subhash C. Minocha

    1996-01-01

    The influence of age of red spruce (Picea rubens Sarg.) cell suspensions on aluminum (Al) effects was studied by adding AICI3 (0.2, 0.5, and 1.0 mM) to the media on each day of a 7-day culture period and analyzing for changes in total cell mass, polyamines, arginine decarboxylase activity, and inorganic ions after 24 h of...

  2. Structural effects of the Azospirillum lipopolysaccharides in cell suspensions.

    Science.gov (United States)

    Matora, L Y; Serebrennikova, O B; Shchyogolev, S Y

    2001-01-01

    The structural influence of Azospirillum lipopolysaccharides (LPS) and lipopolysaccharide-protein complexes (LPPC) on carrot, erythrocyte, and bacterial cell suspensions was explored. The structural potentialities of O-specific polysaccharide fragments of LPS and protein fractions of LPPC were also evaluated. An ability to induce the formation of three kinds of structures in the cell suspensions was revealed depending on the chemical composition of the preparations used. The first and the second ones were connected with effects of cell aggregation (a relatively fast process) and agglutination (a relatively slow process). The third one resulted in phase separation of erythrocyte suspensions (a medium-speed process), with segregating the cells to a separate homogeneous liquid phase.

  3. Development of friable embryogenic callus and embryogenic suspension culture systems in cassava (Manihot esculenta Crantz)

    Science.gov (United States)

    Taylor, N J; Edwards, M; Kiernan, R J; Davey, C D; Blakesley, D; Henshaw, G G

    1996-06-01

    Procedures for the production of a new and highly prolific embryogenic culture system have been developed in cassava. The importance of the basal salts and type of auxin in controlling the development of cassava embryogenic tissues has been demonstrated, with culture on Gresshoff and Doy basal medium in the presence of 4-amino-3,5,6,trichloro-picolinic acid (picloram) inducing the formation of friable embryogenic callus from which highly totipotent embryogenic suspension cultures could be established. Plants have been regenerated from these cultures. The availability of embryogenic suspension cultures is considered to have important implications for the application of genetic transformation and other biotechnologies in the agronomic improvement of cassava.

  4. Cytokine gene expression in murine epidermal cell suspensions: interleukin 1 beta and macrophage inflammatory protein 1 alpha are selectively expressed in Langerhans cells but are differentially regulated in culture.

    Science.gov (United States)

    Heufler, C; Topar, G; Koch, F; Trockenbacher, B; Kämpgen, E; Romani, N; Schuler, G

    1992-10-01

    Epidermal Langerhans cells (LC) are considered direct yet immature precursors of dendritic cells (DC) in the draining lymph nodes. Although the development of LC into potent immunostimulatory DC occurs in vitro and has been studied in detail, little is known about their profile of cytokine gene expression. By using reverse transcriptase polymerase chain reaction analysis to screen 16 cytokines followed by Northern blotting for selected analysis, we determined the cytokine gene expression profile of murine LC at different time points in culture when T cell stimulatory activity is increasing profoundly. LC regularly expressed macrophage inflammatory proteins, MIP-1 alpha and MIP-2, and interleukin 1 beta (IL-1 beta). Both MIPs were downregulated upon culture and maturation into DC, whereas IL-1 beta was strongly upregulated in culture. MIP-1 alpha and IL-1 beta mRNA were found only in LC, but not in other epidermal cells. Apart from trace amounts of IL-6 in cultured LC, several macrophage and T cell products were not detected. The cytokine expression profile of LC thus appears distinct from typical macrophages. The exact role of the cytokine genes we found transcribed in LC remains to be determined.

  5. Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

    Science.gov (United States)

    Stoop, JMH.; Pharr, D. M.

    1993-11-01

    Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures.

  6. Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

    Directory of Open Access Journals (Sweden)

    Sutini Sutini

    2017-02-01

    Full Text Available This study was aimed to assess flavan-3-ol biomass in C. sinensis through callus cultures and suspension cultures derived from leaf explants. Callus initiation of both cultures were using Murashige and Skoog medium were enriched with plant growth regulators Naphtha-lene Acetic Acid 3.0 mg/L and kinetin 2.0 mg/L. The procedures in this study were: (1 callus initiation by cutting the leaves of C. sinen-sis shoots then planted on Murashige and Skoog medium that were enriched with plant growth regulators, (2 sub callus culture on fresh medium that enriched with the same growth regulators, (3 suspension culture initiation of liquid callus, (4 growth examination of callus and suspension cultures in week 12, (5 examination of qualitative-quantitative content of flavan-3-olin suspension cultures at week 4. The results show that suspension cultures contain biomass flavan-3-ol that increase in the same manner of the increase of callus age and weight

  7. Dynamics of indole-3-acetic acid oxidase activity in suspension culture of sunflower crown-gall

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-01-01

    Full Text Available IAA oxidase activity was determined in several growth phases of a suspension culture of sunflower crown-gall. During the short phase of intensive growth (zero passage - PO a negative correlation was noted between enzymatic activity and the rate of growth. IAA oxidase activity increased to a certain level is not a factor limiting cell division. For protraction of the phase of intensive growth (first passage - P1, however, a decrease in the activity of this enzyme seems indispensable. IAA oxidase activity in the tested culture is under the control of inhibitors present in the cells and medium. High enzyme inhibition was observed in PO cells during the phase, of intensive growth and in P1 at the beginning and in the middle part of this phase. These results suggest' that the -auxin level determined in earlier studies in sunflower crown-gall culture is controlled by the IAA oxidase set. During the long phase of intensive growth (P1 this control is of negative feedback type.

  8. Measurements of scattering and absorption in mammalian cell suspensions

    Science.gov (United States)

    Mourant, Judith R.; Freyer, James P.; Johnson, Tamara M.

    1996-04-01

    During the past several years a range of spectroscopies, including fluorescence and elastic- scatter spectroscopy, have been investigated for optically based detection of cancer and other tissue pathologies. Both elastic-scatter and fluorescence signals depend, in part, on scattering and absorption properties of the cells in the tissue. Therefore an understanding of the scattering and absorption properties of cells is a necessary prerequisite for understanding and developing these techniques. Cell suspensions provide a simple model with which to begin studying the absorption and scattering properties of cells. In this study we have made preliminary measurements of the scattering and absorption properties of suspensions of mouse mammary carcinoma cells (EMT6) over a broad wavelength range (380 nm to 800 nm).

  9. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

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

  10. Production of secondary metabolites trimethyl xanthina by Camellia sinensis L suspension culture

    Science.gov (United States)

    Sutini, Sodiq, Mochamad; Muslihatin, Wirdhatul; Indra, Mochamad Rasjad

    2017-06-01

    Bioactive trimethyl xanthina can be obtained from the plant Camellia sinensis L. To obtain bioactive plant of which there are several hurdles for instance to wait up to five years to be harvested, also it needs land at a certain height from the sea level. Therefore, the production of secondary metabolites trimethyl xanthina need to be developed with suspense culture techniques. The purpose of this study obtained the production of bioactive trimethyl xanthina way culturally suspense in large scale with a relatively short time, potentially as anti-oxidants. Research methods include: (1) initiation of callus from pieces of leaves, shoots the youngest of the plant Camellia sinensis L in the media MS with the optimization of the addition of growth regulators, (2) the subculture of callus on media and plant growth regulator that is equal to the stage of initiation, (3) initiation of suspension culture using explants of callus Camellia sinensis L, (4) Analysis of secondary metabolites trimethyl xanthina growth in suspension culture, (5) the isolation and identification of trimethyl xanthina qualitatively and quantitatively using thin layer chromatography/high performance chromatography column. The results of the study suspension cultures containing bioactive trimethyl xanthina candidates that can be used as an antioxidant.

  11. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Directory of Open Access Journals (Sweden)

    Priscila Cardoso Cristovam

    2008-10-01

    Full Text Available OBJETIVO: Avaliar a importância da presença de células 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo obtido de rimas córneo-esclerais. MÉTODOS: Rimas de diferentes doadores tiveram seus estroma posterior e endotélio removidos (n=6. Cada rima foi dividida em três segmentos iguais, que foram colocados em cultura em três diferentes condições: um segmento foi colocado na placa de cultura com o lado epitelial para cima (Grupo A. Os dois segmentos restantes foram tripsinizados e a suspensão de células obtida foi cultivada com (Grupo B ou sem (Grupo C células 3T3 irradiadas. As células foram mantidas em meio de cultura "supplemental hormonal epithelial médium" (SHEM, a migração epitelial e a formação de clones nos grupos A, B e C foram avaliadas pela microscopia de contraste de fase e por coloração pela rodamina B. Os resultados foram comparados estatisticamente. RESULTADOS: O crescimento de células epiteliais foi observado em 4/6 rimas (Grupo A. Todas as suspensões de células epiteliais que foram cultivadas com células 3T3 (Grupo B formaram clones. Nenhuma adesão ou formação de clones verdadeiros (holo ou meroclones foi observada na cultura de células que foi cultivada sem 3T3 (Grupo C (p=0,009. CONCLUSÕES: Suspensão de células epiteliais límbicas obtidas de rimas córneo-esclerais no modelo utilizado precisa ser cultivada com células 3T3 para formar clones e estabelecer colônias epiteliais com perspectivas para uso terapêutico na reconstrução da superfície ocular.PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR. METHODS: Corneo-scleral rims from different donors (n=6 had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the

  12. Assessment of drug salt release from solutions, suspensions and in situ suspensions using a rotating dialysis cell

    DEFF Research Database (Denmark)

    Parshad, Henrik; Frydenvang, Karla; Liljefors, Tommy

    2003-01-01

    buffer is used as release media. Generally, the initial release of the drug salt from in situ suspensions occurred faster as compared to conventional suspensions, probably due to incomplete precipitation of the drug salt, and hence formation of supersaturated solutions where the rate of release......A rotating dialysis cell consisting of a small (10 ml) and a large compartment (1000 ml) was used to study the release of drug salt (bupivacaine 9-anthracene carboxylate) from (i). solutions, (ii). suspensions and (iii). in situ formed suspensions. Initial release experiments from suspensions...... indicated that the release of drug salt in deionized water was predominantly limited by the diffusion across the membrane whereas it is essentially dissolution rate controlled in 0.05 M phosphate buffer (pH 7.40). Thus, the in vitro model appears to have a potential in formulation screening when phosphate...

  13. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  14. Growth, purification and characterization of Semliki Forest virus in Ehrlich ascites tumor cell suspensions.

    Science.gov (United States)

    Ivanic, S

    1976-01-01

    The growth of Semliki Forest Virus (SFV) in suspension cultures of Ehrlich Ascites (EA) cells and its purification is described. Large volumes of virus material were concentrated by filtration with DIAFLO XM-300 membrane and precipitation with ammonium sulfate. A combination of protamine sulfate treatment, centrifugation of the virus onto a 50 per cent sucrose cushion, and sedimentation through a 5--30 per cent sucrose density gradient was employed. The purified virus particles were homogeneous as revealed by electron microscopy, by moving boundary electrophoresis, and by polyacrylamide gel electrophoresis. Virus suspensions containing 1 mg/ml of protein had a hemagglutinin titer of 1:12,000 when measured with 0.25 per cent goose red blood cells.

  15. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

    DEFF Research Database (Denmark)

    de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F

    2014-01-01

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon...... polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model...... plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium...

  16. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : IV. Induction of Vanillic Acid Formation.

    Science.gov (United States)

    Funk, C; Brodelius, P E

    1992-05-01

    Kinetin is used as an elicitor to induce vanillic acid formation in cell suspension cultures of Vanilla planifolia. Maximal induction is observed at a kinetin concentration of 20 micrograms per gram of fresh weight of cells. Vanillic acid synthesis is observed a few hours after elicitation. The effects of kinetin on the activity of some enzymes of the phenylpropanoid pathway, i.e. phenylalanine ammonia-lyase, 4-hydroxycinnamate:coenzyme A ligase and uridine 5'-diphosphate-glucose:trans-cinnamic acid glucosyltransferase, are reported and compared to the effects of chitosan. The former two enzymes are induced by chitosan with a maximum activity of approximately 25 to 40 hours after elicitation. All three enzymes are induced by kinetin with maximum activities for phenylalanine ammonia lyase and 4-hydroxycinnamate:coenzyme A ligase at approximately 50 hours after induction, whereas maximum glucosyltransferase activity is seen already after 24 hours. Furthermore, both elicitors induced the formation of lignin-like material, whereas only kinetin induced vanillic acid biosynthesis. Finally, kinetin but not chitosan induces catechol-4-O-methyltransferase activity, catalyzing the formation of 4-methoxycinnamic acids, which were shown to be intermediates of hydroxybenzoic acid biosynthesis within cells of V. planifolia. It is suggested that this methyltransferase is directly involved in the biosynthesis of vanillic acid.

  17. Basic cell culture.

    Science.gov (United States)

    Pollard, J W

    1990-01-01

    This article will describe the basic techniques required for successful cell culture. It will also act to introduce some of the other chapters in this volume. It is not intended, as this volume is not, to describe the establishment of a tissue culture laboratory, nor to provide a historical or theoretical survey of cell culture. There are several books that adequately cover these areas, including the now somewhat dated but still valuable volume by Paul (1), the multi-authored Methods in Enzymology volume edited by Jakoby and Pastan (2), and the new edition of Freshney (3). Instead, this chapter's focus will be on the techniques for establishing primary rodent cell cultures from embryos and adult skin, maintaining and subculturing these fibro-blasts and their transformed derivatives, and the isolation of genetically pure strains. The cells described are all derived from Chinese hamsters since, to date, these cells, have proved to be the most useful for somatic cell genetics (4,5). The techniques, however, are generally applicable to most fibroblastic cell types.

  18. Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

    Science.gov (United States)

    Moreira, J L; Alves, P M; Rodrigues, J M; Cruz, P E; Aunins, J G; Carrondo, M J

    1994-11-30

    Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

  19. Separation of Beating Cardiac Myocytes from Suspensions of Heart Cells

    Science.gov (United States)

    Pretlow, Thomas G.; Glick, Melvin R.; Reddy, William J.

    1972-01-01

    Heart cells were obtained in suspension after incubation with collagenase and hyaluronidase in Saline A. Cardiac myocytes were separated by isopycnic centrifugation in 88.6 to 92.4% purity from other heart cells with different densities, and by velocity or rate-zonal sedimentation, in 92.8 to 97.4% purity from heart cells with different diameters. A previously described computer integration of the differential sedimentation equation was used to determine the centrifugal force, duration of centrifugation and gradient design, which would permit the separation of cardiac myocytes from other heart cells by velocity sedimentation. The myocytes continued to contract rhythmically after being recovered from the density gradients. Velocity sedimentation was superior to isopycnic sedimentation for the separation of cardiac myocytes from heart cell suspensions because it gave the most highly purified myocytes, resulted in recovery of the largest proportion of myocytes in purified fractions from the gradient and required lower centrifugal forces for shorter periods of time. The potential significance of the availability of pure cardiac myocytes is discsused. ImagesFig 2Fig 1 PMID:4336547

  20. [Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

    Science.gov (United States)

    Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

    2015-01-01

    The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

  1. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

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

  2. Signal transduction events in aluminum-induced cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Woltering, E.J.

    2007-01-01

    In this study, some of the signal transduction events involved in AlCl3-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 ¿M AlCl3 showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation.

  3. Induction and Analysis of the Alkaloid Mitragynine Content of a Mitragyna speciosa Suspension Culture System upon Elicitation and Precursor Feeding

    Directory of Open Access Journals (Sweden)

    Nor Nahazima Mohamad Zuldin

    2013-01-01

    Full Text Available This study aimed to determine the effects of different concentrations and combinations of the phytohormones 2,4-dichlorophenoxy acetic acid (2,4-D, kinetin, 6-benzylaminopurine (BAP, and 1-naphthaleneacetic acid (NAA on callus induction and to demonstrate the role of elicitors and exogenous precursors on the production of mitragynine in a Mitragyna speciosa suspension culture. The best callus induction was achieved from petiole explants cultured on WPM that was supplemented with 4 mg L−1 2, 4-D (70.83%. Calli were transferred to liquid media and agitated on rotary shakers to establish Mitragyna speciosa cell suspension cultures. The optimum settled cell volume was achieved in the presence of WPM that contained 3 mg L−1 2,4-D and 3% sucrose (9.47±0.4667 mL. The treatment of cultures with different concentrations of yeast extract and salicylic acid for different inoculation periods revealed that the highest mitragynine content as determined by HPLC was achieved from the culture treated with 250 mg L−1 yeast extract (9.275±0.082 mg L−1 that was harvested on day 6 of culturing; salicylic acid showed low mitragynine content in all concentrations used. Tryptophan and loganin were used as exogenous precursors; the highest level of mitragynine production was achieved in cultures treated with 3 μM tryptophan and harvested at 6 days (13.226±1.98 mg L−1.

  4. Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, Hiroko; Seto, Akira

    1981-01-01

    A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

  5. Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

    Science.gov (United States)

    Hartmann, S; Lang, H; Reuther, G

    1992-10-01

    Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2-3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green "torpedo"-shaped somatic embryos after 6-8 weeks from protoplast isolation.

  6. Convection in a colloidal suspension in a closed horizontal cell

    International Nuclear Information System (INIS)

    Smorodin, B. L.; Cherepanov, I. N.

    2015-01-01

    The experimentally detected [1] oscillatory regimes of convection in a colloidal suspension of nanoparticles with a large anomalous thermal diffusivity in a closed horizontal cell heated from below have been simulated numerically. The concentration inhomogeneity near the vertical cavity boundaries arising from the interaction of thermal-diffusion separation and convective mixing has been proven to serve as a source of oscillatory regimes (traveling waves). The dependence of the Rayleigh number at the boundary of existence of the traveling-wave regime on the aspect ratio of the closed cavity has been established. The spatial characteristics of the emerging traveling waves have been determined

  7. Changes in auxin level in the course of growth of a sunflower crown-gall suspension culture

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-01-01

    Full Text Available The auxin level in the cell mass and culture medium was determined by means of the Avena straight caleoptile test in various periods of the suspension culture cycle of the sunflower crown-gall tumour. The investigations were performed in the course of the zero passage (PO and first one (Pl, differing in their time of duration of maximum growth and its intensity. In both passages the intra- and extra-cellular auxin levels reach values of the same order. At the beginning of the maximal growth phase the activity corresponding to IAA in the cells prevails over that of the other auxin-like compounds. This disproportion diminishes with further development of the culture, and with the beginning of the stationary phase the cellular IAA level is lower than that of the remaining auxin-like compounds. The short phase of maximal growth (PO occurs with an auxin level decreasing in the cell mass and increasing in the medium, and towards the end of the cycle these levels become equal. During the long phase of maximal growth (Pl the total amount of auxins in the cells increases and is 2-3 times higher than in the medium, whereas IAA in the cells remains at a constant level. These results suggest that the participation of IAA in the intracellular pool of auxin-like substances is decisive for the mitotic activity of the cells and maintenance of growth in the culture.

  8. Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement

    NARCIS (Netherlands)

    le Gac, Severine; Zwaan, Ed; van den Berg, Albert; Ohl, C.D.

    2007-01-01

    We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells

  9. Stereotaxic implantation of dispersed cell suspensions into brain. A systematic appraisal of cell placement and survival

    International Nuclear Information System (INIS)

    Plunkett, R.J.; Weber, R.J.; Oldfield, E.H.

    1988-01-01

    The application of several recent advances in cell biology, brain implantation, and cell-mediated tumor immunotherapy requires successful and reproducible placement of viable cell suspensions into brain. Stereotaxic implantation is being used to inject cytotoxic lymphocytes into gliomas and to replace dopaminergic cells in parkinsonian models. Systematic assessment of the factors that influence success in implantation of cell suspensions into solid tissues is needed. A model was developed for investigation of stereotaxic implantation using radiolabeled rat lymphokine-activated killer (LAK) cells. Anesthetized rats received microliter injections of cell suspension into the right caudate nucleus. The injection volume, cell concentration, infusion rate, and needle size were varied systematically. The animals were sacrificed 1 hour after injection; the brain was removed and sectioned, and the radioactivity was counted. Three aliquots of the suspension were injected into counting tubes for control analysis. Recovery of radioactivity was expressed as the percent of mean counts per minute (cpm) in the right frontal lobe/mean cpm in the three control tubes. To assess the viability of implanted cells, the right frontal region was mechanically dissociated in media and centrifuged, and the pellet and supernatant were counted. By using small needles and slow infusion of volumes of 10 microliters or less, 85% to 90% of the radioactivity was recovered in the caudate nucleus. At least half of the implanted cells were viable. Consistent, accurate implantation of dispersed cells into brain over a range of volumes, cell concentrations, infusion rates, and needle sizes was achieved

  10. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  11. Establishment of Aquilaria malaccensis Callus, cell suspension and adventitious root systems

    International Nuclear Information System (INIS)

    Norazlina Noordin; Rusli Ibrahim

    2010-01-01

    Aquilaria malaccensis is a tropical forest tree from the family Thymelaeaceae, an endangered forest species and was listed in CITES since 1995. Locally known as Pokok Karas, this tree produces agar wood or gaharu, a highly valuable, resinous and fragrant forest product. Karas has been highly recognized for its vast medicinal values and gaharu has been widely use for perfumery, incense and religious purposes. The phyto chemical studies of agar wood showed that Sesqui terpenoid and Phenyl ethy chromone derivatives are the principal compounds that have anti allergic and anti microbe activities. Cell and organ culture systems provide large scale production of biomass and offers feasibilities for the production of secondary metabolites. This paper describes the work done for establishing reproducible systems for callus initiation and production of cell suspension cultures as well as production of adventitious roots that will later be amenable for the production of secondary metabolites of A. malaccensis. Hence, further manipulation with Methyl Jasmonate, a chemical elicitor could be done to induce secondary metabolites using callus, cell suspension and adventitious roots systems. (author)

  12. Stimulation of taxane production in suspension cultures of Taxus ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-06-17

    Jun 17, 2008 ... Planta 215: 596-605. Nothnagel EA, McNeil M, Albersheim P, Dell A (1983). Host pathogen interaction XXII: a galacturonic acid oligosaccharide from plant cell wall elicits phytoalexins. Plant Physiol. 71: 916-926. Ramesh NP (1998). Tour de paclitaxel: biocatalysis for semisynthesis. Annu. Rev. Microbiol.

  13. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  14. Influence of suspension osmolarity and erythrocyte volume on cell deformability

    Energy Technology Data Exchange (ETDEWEB)

    Feo, C. (Institut de Pathologie Cellulaire, INSERM, 94 - Kremlin-Bicetre (France)); Phillips, W.M. (School of Mechanical Engineering, Purdue University, West Lafayette, IN (USA))

    1982-01-01

    Erythrocytes were suspended in dextran solutions of phosphate buffered saline with solution osmolarities from 400 to 20 mosM/kg. The dilute suspensions were subjected to linear shear and their deformation determined by laser diffractometry (Ektacytometer). Cell volumes were measured using a Coulter counter following fixation in glutaraldehyde to eliminate the influence of deformability on the volume measurement. Minimum deformability generally agreed with the maximum cellular volume produced by hypotonic solutions. However, reduced deformability was observed for both hyperosmotic and hypoosmotic conditions. The oncotic effect of the dextran delayed hemolysis to surprisingly low values of solution osmolarity. In contrast with the usual osmotic fragility results, in the hypotonic dextran solutions there was no evidence of hemoglobin release. At low shear stresses, deformability was found to be enhanced by reducing intracellular viscosity (via osmotic water transport into the cell). However, the maximum cellular deformation obtained at high shear stress was always less than for the normal discocyte at normal osmolarities.

  15. Microfluidic Cell Culture Device

    Science.gov (United States)

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

    2014-01-01

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

  16. Production of saponins from Panax ginseng suspension and adventitious root cultures

    Czech Academy of Sciences Publication Activity Database

    Langhansová, Lenka; Maršík, Petr; Vaněk, Tomáš

    2005-01-01

    Roč. 49, č. 3 (2005), s. 463-465 ISSN 0006-3134 R&D Projects: GA ČR(CZ) GP521/02/P064; GA MŠk(CZ) OC 843.10; GA MŠk(CZ) ME 671 Institutional research plan: CEZ:AV0Z4055905 Keywords : suspension cultures * bioreactor * ginsenosides Subject RIV: EI - Biotechnology ; Bionics Impact factor: 0.792, year: 2005

  17. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

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

  18. Suspended Cell Culture ANalysis (SCAN) Tool to Enhance ISS On-Orbit Capabilities, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences and partner, Draper Laboratory, propose to develop an on-orbit immuno-based label-free Suspension Cell Culture ANalysis tool, SCAN tool, which...

  19. Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.).

    Science.gov (United States)

    Yang, Jun; Bi, Hui-Ping; Fan, Wei-Juan; Zhang, Min; Wang, Hong-Xia; Zhang, Peng

    2011-12-01

    Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l(-1) 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l(-1) hygromycin and 200 mg l(-1) cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  20. Enrichment of unlabeled human Langerhans cells from epidermal cell suspensions by discontinuous density gradient centrifugation

    NARCIS (Netherlands)

    Teunissen, M. B.; Wormmeester, J.; Kapsenberg, M. L.; Bos, J. D.

    1988-01-01

    In this report we introduce an alternative procedure for enrichment of human epidermal Langerhans cells (LC) from epidermal cell suspensions of normal skin. By means of discontinuous Ficoll-Metrizoate density gradient centrifugation, a fraction containing high numbers of viable, more than 80% pure

  1. Panax ginseng Adventitious Root Suspension Culture: Protocol for Biomass Production and Analysis of Ginsenosides by High Pressure Liquid Chromatography.

    Science.gov (United States)

    Murthy, Hosakatte Niranjana; Paek, Kee Yoeup

    2016-01-01

    Panax ginseng C.A. Meyer (Korean ginseng) is a popular herbal medicine. It has been used in Chinese and Oriental medicines since thousands of years. Ginseng products are generally used as a tonic and an adaptogen to resist the adverse influence of a wide range of physical, chemical and biological factors, and to restore homeostasis. Ginsenosides or ginseng saponins are the principal active ingredients of ginseng. Since ginseng cultivation process is very slow and needs specific environment for field cultivation, cell and tissue cultures are sought as alternatives for the production of ginseng biomass and bioactive compounds. In this chapter, we focus on methods of induction of adventitious roots from ginseng roots, establishment of adventitious root suspension cultures using bioreactors, procedures for processing of adventitious roots, and analysis of ginsenosides by high pressure liquid chromatography.

  2. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...... a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment....

  3. Cell culture supernatants for detection perforin ELISA

    African Journals Online (AJOL)

    Najwa

    2014-02-19

    Feb 19, 2014 ... (2001). The lymphocytes were isolated from the peripheral heap- rinized whole blood as follows: 3 ml of blood was centrifuged at. 1000 rpm for 15 min, buffy coat was collected in a 10 ml centrifuge tubes and diluted with 5 ml RPMI 1640 (cell suspension), 5 ml of the diluted cell suspension was layered on 3 ...

  4. Understanding Romanowsky staining. 2. The staining mechanism of suspension-fixed cells, including influences of specimen morphology on the Romanowsky-Giemsa effect.

    Science.gov (United States)

    Horobin, R W; Curtis, D; Pindar, L

    1989-01-01

    Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.

  5. Antioxidant isoenzyme responses to nickel-induced stress in tobacco cell suspension culture Resposta de isoenzimas antioxidantes ao estresse induzido por níquel em cultura de células em suspensão de fumo

    Directory of Open Access Journals (Sweden)

    Georgia Bertoni Pompeu

    2008-01-01

    Full Text Available Exposure to nickel (Ni at high concentrations can lead to production of reactive oxygen species (ROS resulting in oxidative damage at the cellular level. We investigated the antioxidative responses of Nicotiana tabacum cv BY-2 cell suspension to Ni stress (0.075 and 0.75 mM NiCl2 over a 72 h period with special attention to potential alterations in isoenzymes of superoxide dismutase (SOD, catalase (CAT and glutathione reductase (GR. Two main SOD isoenzymes were observed, a Mn-SOD (band I and a Fe-SOD (band II, as well as one CAT isoenzyme and four GR isoenzymes. Activity staining analysis revealed that CAT activity plays a major role in the early response to Ni-induced oxidative stress, particularly when the Ni concentration used was low, whilst a specific GR isoenzyme appears to respond to the Ni-induced oxidative stress when a much higher Ni concentration was used to induce the stress for the same period of treatment. These results illustrate the importance and advantages of determining individual isoenzyme activities.A exposição ao níquel (Ni, em altas concentrações, pode levar à produção de espécies reativas de oxigênio (EAOs, resultando em danos oxidativos em nível celular. Foram investigadas as respostas antioxidativas de células em suspensão do cultivar BY-2 de Nicotiana tabacum submetidas ao estresse por Ni (0.075 e 0.75 mM de NiCl2 por 72 h, com atenção especial às alterações potencias em isoenzimas de superóxido dismutase (SOD, catalase (CAT e glutatione redutase (GR. Duas principais isoenzimas de SOD foram observadas, uma Mn-SOD (banda I e outra Fe-SOD (banda II, bem como uma isoenzima CAT e quatro isoenzimas de GR. As análises revelaram que a atividade de CAT tem papel principal no momento inicial de resposta ao estresse oxidativo induzido por Ni, particularmente, quando sua concentração foi mais baixa, enquanto uma isoenzima específica de GR parece responder a este estresse na concentração mais alta de Ni

  6. Generation of highly productive polyclonal and monoclonal tobacco suspension lines from a heterogeneous transgenic BY-2 culture through flow cytometric sorting

    OpenAIRE

    Kirchhoff, Janina

    2012-01-01

    Over the last 20 years, plant cells gained increasing interest as host systems for the production of human and animal pharmaceuticals. Transgenic tobacco suspension cultures are an especially promising production host because of their beneficial growth, low maintenance requirement and the possibility of contained cultivation in bioreactors under controlled conditions. The excellent product quality of plant-produced proteins was demonstrated for a multitude of proteins, but the heterogeneous a...

  7. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

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

  8. The effects of cellulase on capsaicin production in freely suspended cells and immobilized cell cultures of capsicum annuum

    International Nuclear Information System (INIS)

    Islek, C.

    2014-01-01

    The effect of different concentrations of cellulase on the production of capsaicin in freely suspended cell and immobilized cell cultures of Kahramanmara pepper seeds (Capsicum annuum L.) were studied. Calluses were obtained from in vitro germinated hypocotyl explants of pepper seedlings and cell suspensions were prepared from these calluses. Immobilized cell suspension cultures with calcium alginate and free cell suspension cultures were obtained by using cell suspensions. Elicitor such as cellulase (5-30 micro g/ml), was applied both for the free and immobilized cell suspensions and control group without elicitor was prepared. The concentration of capsaicin in freely suspended cells, immobilized cells and their filtrates were identified by HPLC after extraction with ethyl acetate. It was found that the immobilization process had an increasing effect on the capsaicin accumulation. The concentration of capsaicin in the immobilized cells for both control groups and elicitor added samples was higher than the free cells. In general, capsaicin concentration in the filtrate for free cells was higher than the immobilized cells. When all the cellulase and the sampling hours were compared, the highest capsaicin concentration for the immobilized cells was determined as 362,91 micro g/ml f.w. at the 24th hour for 30 micro g/ml cellulase applied samples. (author)

  9. Proteomic analysis of grape berry cell cultures reveals that developmentally regulated ripening related processes can be studied using cultured cells.

    Directory of Open Access Journals (Sweden)

    Ramaschandra G Sharathchandra

    Full Text Available BACKGROUND: This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. CONCLUSIONS: The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks

  10. Ethylene signaling in salt stress- and salicylic acid-induced programmed cell death in tomato suspension cells.

    Science.gov (United States)

    Poór, Péter; Kovács, Judit; Szopkó, Dóra; Tari, Irma

    2013-02-01

    Salt stress- and salicylic acid (SA)-induced cell death can be activated by various signaling pathways including ethylene (ET) signaling in intact tomato plants. In tomato suspension cultures, a treatment with 250 mM NaCl increased the production of reactive oxygen species (ROS), nitric oxide (NO), and ET. The 10(-3) M SA-induced cell death was also accompanied by ROS and NO production, but ET emanation, the most characteristic difference between the two cell death programs, did not change. ET synthesis was enhanced by addition of ET precursor 1-aminocyclopropane-1-carboxylic acid, which, after 2 h, increased the ROS production in the case of both stressors and accelerated cell death under salt stress. However, it did not change the viability and NO levels in SA-treated samples. The effect of ET induced by salt stress could be blocked with silver thiosulfate (STS), an inhibitor of ET action. STS reduced the death of cells which is in accordance with the decrease in ROS production of cells exposed to high salinity. Unexpectedly, application of STS together with SA resulted in increasing ROS and reduced NO accumulation which led to a faster cell death. NaCl- and SA-induced cell death was blocked by Ca(2+) chelator EGTA and calmodulin inhibitor W-7, or with the inhibitors of ROS. The inhibitor of MAPKs, PD98059, and the cysteine protease inhibitor E-64 reduced cell death in both cases. These results show that NaCl induces cell death mainly by ET-induced ROS production, but ROS generated by SA was not controlled by ET in tomato cell suspension.

  11. A numerical model of localized convection cells of Euglena suspensions

    Science.gov (United States)

    Iima, Makoto; Shoji, Erika; Yamaguchi, Takayuki

    2014-11-01

    Suspension of Euglena gracilis shows localized convection cells when it is illuminated form below with strong light intensity. Experiments in an annular container shows that there are two elementary localized structures. One consists of a pair of convection cells and a single region where number density of Euglena is high. The other consists a localized traveling wave. Based on the measurements of the flux of number density, we propose a model of bioconvection incorporating lateral phototaxis effect proportional to the light intensity gradient. Using pseudo spectral method, we performed numerical simulation of this model. We succeed in reproducing one of the localized structures, a convection pair with single region of high number density. Also, when the aspect ratio is large, there are a parameter region where the localized structure and conductive state are both stable, which is suggested by experiments. Spatial distribution of the number density implies that the accumulation of microorganism due to the convective flow causes such bistability. CREST(PJ74100011) and KAKENHI(26400396).

  12. The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application

    DEFF Research Database (Denmark)

    Dong, Jing; Bowra, Steve; Vincze, Éva

    2010-01-01

    Background The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell...... suspension culture from both species. Results We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we...... level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots....

  13. [Effect of precursors on the production of coumarins in a suspension culture of Angelica archangelica L].

    Science.gov (United States)

    Siatka, T; Kasparová, M

    2002-01-01

    The effect of potential precursors (cinnamic acid, phenylalanine, tyrosine) in three concentrations (0.01; 0.1; 1 mmol/l) on the growth of the culture and coumarin production was investigated in the suspension culture of Angelica archangelica L. The cultures were cultivated under constant illumination (3500 lux) and in the dark. The growth of the culture in the dark was not affected by the precursors, cinnamic acid in a concentration of 1 mmol/l exerted toxic action. The growth of the culture cultivated under constant illumination was stimulated by tyrosine in a concentration of 0.1 and 1 mmol/l, and phenylalanine in a concentration of 0.1 mmol/l; phenylalanine and cinnamic acid in a concentration of 1 mmol/l inhibited the growth of the culture. In the cultivation in the dark, coumarin production was increased by phenylalanine in a concentration 0.01 mmol/l and by tyrosine in a concentration of 1 mmol/l. In the cultivation under constant illumination, coumarin production was decreased by the action of cinnamic acid, was not affected by tyrosine, and phenylalanine (0.01 and 0.1 mmol/l) increased it in comparison with the culture without precursors.

  14. [Fluid suspension co-culture of Dendrobium officinale protocorm and living fungus].

    Science.gov (United States)

    Meng, Zhixia; Shu, Ying; Wang, Chunlan; Guo, Shunxing

    2012-06-01

    To set up a system for fluid suspension co-culture of Dendrobium officinale protocorm and living fungus MF24, so as to provide certain scientific evidence for industrial production of protocorm. Whether the protocorm culture system was suitable for the normal growth of MF24 fungus were studied, the growth of protocorm cultured alone and co-cultured with the fungus were researched under light and dark culture conditions, the biomass and proliferation times were determined, and HPLC method was used to analyze and compare the changes of 11 characteristic peak areas in D. officinale protocorm. The MF24 fungus could grow normally in the 6,7-V liquid medium used to culture the protocorm, and when it was cultured by 8-10 hours per day under 1 500 lx, the growth rate of the fungus was slowed. Protocorm could grow normally in light and dark culture conditions, and add the MF24 fungus in the early cultivation stages of protocorm, both inhibit the growth of each other. In the protocorm for the growth stability to add 5 diameter 9 mm fungi block, the protocorm growth and chemical composition type had no significant effect. However, under illumination, co-cultured for 5 days protocorm of which 10 compounds content decreased 13.64% to 138.47%, in dark conditions, co-cultured for 5 days protocorm of which 7 compounds increased by 0.71% to 12.82%, and 4 compounds slightly reduced by 3.03% to 14. 14% compared with the control. Under the appropriate condition, living fungus MF24 could co-culture with the D. officinale protocorm, and affected the latter's secondary metabolite levels.

  15. Growth characteristics and nutrient depletion of Miscanthus x ogiformis Honda 'Giganteus' suspension cultures

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted

    1998-01-01

    The growth characteristics and nutrient depletion in suspension cultures of Miscanthus ogiformis Honda ‘Giganteus' grown in media containing either Murashige and Skoog or N6 basal nutrient salts were studied during a culture period of 15 days. Proline was added to both media in concentrations from...... 0 to 300 mM. The fresh and dry weights of the suspension aggregates and the concentrations of ammonium, nitrate, proline and sugar remaining in the medium were measured at different points in time during the culture period. The results showed an almost total depletion of ammonium but a limited...

  16. Evaluation of Antioxidant and Antibacterial Potentials of Nigella sativa L. Suspension Cultures under Elicitation

    Directory of Open Access Journals (Sweden)

    Hera Chaudhry

    2015-01-01

    Full Text Available Nigella sativa L. (family Ranunculaceae is an annual herb of immense medicinal properties because of its major active components (i.e., thymoquinone (TQ, thymohydroquinone (THQ, and thymol (THY. Plant tissue culture techniques like elicitation, Agrobacterium mediated transformation, hairy root culture, and so on, are applied for substantial metabolite production. This study enumerates the antibacterial and antioxidant potentials of N. sativa epicotyl suspension cultures under biotic and abiotic elicitation along with concentration optimization of the elicitors for enhanced TQ and THY production. Cultures under different concentrations of pectin and manganese chloride (MnCl2 elicitation (i.e., 5 mg/L, 10 mg/L, and 15 mg/L showed that the control, MnCl2 10 mg/L, and pectin 15 mg/L suspension extracts greatly inhibited the growth of E. coli, S. typhimurium, and S. aureus (MIC against E. coli, i.e., 2.35±0.8, 2.4±0.2, and 2.46±0.5, resp.. Elicitation decreased SOD enzyme activity whereas CAT enzyme activity increased remarkably under MnCl2 elicitation. MnCl2 10 mg/L and pectin 15 mg/L elicitation enhanced the DPPH radical inhibition ability, but ferric scavenging activity was comparable to the control. TQ and THY were quantified by LC-MS/MS in the cultures with high bioactive properties revealing maximum content under MnCl2 10 mg/L elicitation. Therefore, MnCl2 elicitation can be undertaken on large scale for sustainable metabolite production.

  17. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : II. Effects of Precursor Feeding and Metabolic Inhibitors.

    Science.gov (United States)

    Funk, C; Brodelius, P E

    1990-09-01

    Feeding of cinnamic acid and ferulic acid to non-treated and chitosan-treated cell suspension cultures of Vanilla planifolia resulted in the formation of trace amounts of p-hydroxy benzoic acid (5.2 micrograms per gram fresh weight of cells) and vanillic acid (6.4 micrograms per gram fresh weight of cells), respectively. Addition of a 4-hydroxycinnamate: CoA-ligase inhibitor, 3,4-(methylenedioxy)-cinnamic acid (MDCA), resulted in a reduced biosynthesis of ligneous material with a simultaneous significant increased vanillic acid formation (around 75 micrograms per gram fresh weight of cells). A K(1) of 100 micromolar for 4-hydroxycinnamate: CoA-ligase in a crude preparation was estimated for this inhibitor. It is suggested that the conversion of cinnamic acids into benzoic acids does not involve cinnamoyl CoA esters as intermediates. Feeding of (14)C-cinnamic acid and (14)C-ferulic acid to cells treated with MDCA indicate that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of V. planifolia.

  18. Evaluation of Antioxidant Enzyme Activity and Biochemical Characterization from Static and Suspension Culture of Withania somnifera L.

    Directory of Open Access Journals (Sweden)

    Satyajit Kanungo

    2015-03-01

    Full Text Available Withania somnifera (L. Dunal, is an erect evergreen shrub commonly known as Ashwagandha. It is widely used in Ayurvedic and in the traditional pharmacopeia system of India. It is one of the major ingredients in many formulations prescribed for a variety of musculoskeletal conditions including arthritis and rheumatism. In the present study the variation in quality and quantity of protein and antioxidant enzymes were evaluated biochemically and enzymatically from the static and suspension cultures. The nodal segments had provided maximum callusing of 90.25±0.06 % with (1mg/l of BAP and Kn with (2mg/l of 2, 4-D. The static and suspension cultures were taken for the analysis of total soluble protein and screened for antioxidant enzyme activity [catalase (CAT, superoxide dismutase (SOD and guaiacol peroxidase (GPX]. The protein content (1.2016 µg/µl was found to be higher in static culture samples (0.870 µg/µl than the protein obtained from the suspension culture. The antioxidant enzyme activity (CAT, SOD and GPX was higher in static culture samples (301.01± 0.42, 198.92 ± 0.29, 103.75 ± 0.11 nkat/ mg of protein than that of suspension culture. Specific activity staining of isozyme pattern exhibited three isoforms (CAT 1, CAT 2 and CAT 3 in static culture samples but CAT 1 was absent in the sample extracted from suspension cultures.  In case of SOD, four bands (SOD 1, SOD 2, SOD 3 and SOD 4 were found in both the samples whereas intensity of GPX activity was found to be more in static culture but both the samples exhibited three isoforms such as  (GPX 1, GPX 2 and GPX 3. The supplementation of required nutrients along with the phytohormones under in vitro condition might be an enhancing factor to yield antioxidant enzymes in the static culture samples. 

  19. Ureaplasma infection of cell cultures.

    Science.gov (United States)

    Kotani, H; McGarrity, G J

    1986-05-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.

  20. Cell suspension concentration monitoring by using a miniaturized serial high frequency SAWR sensor

    OpenAIRE

    Li, Jian; Feng, Hailin; Fang, Yiming

    2015-01-01

    In this paper, a miniaturized cell suspension concentration monitoring method was investigated. The sensing unit was a carbon screen-printed electrode (CSPE) in serial with a 433MHz vacuum-packaged surface acoustic wave resonator (SAWR). SAWR provided a stable and high operating frequency, which helps to keep the stability and sensitivity of the monitoring system. Living cells suspensions in different concentrations were prepared and dropcast on CSPE. Frequency responses of the sensor were re...

  1. Further characterization of the adhesive-tumor-cell culture system for measuring the radiosensitivity of human tumor primary cultures

    International Nuclear Information System (INIS)

    Brock, W.A.; Bock, S.P.; Williams, M.; Baker, F.L.

    1987-01-01

    This study extends the use of the adhesive-tumor-cell culture system to include: over 100 sensitivity measurements at 2.0 Gy; tumorgenicity determinations in nude mice; and flow cytometry of the cells grown in the system. The malignant nature of the growing cells was proved by injecting cells into nude mice. Tumors resulted in 60% of the cases and the histology of each xenograft was similar to that of the human tumor. Flow cytometry was used to obtain DNA histograms of the original cell suspension and of cultures during the two week culture period in order to obtain quantitative information about the growth of aneuploid versus diploid populations. The results thus far demonstrate that 95% of aneuploid populations yield aneuploid growth; of the first 20 cases studied, only one suspension with an aneuploid peak resulted in diploid growth. Of further interest was the observation that it is not unusual for a minor aneuploid population to become the predominate growth fraction after two weeks in culture. These results demonstrate that the adhesive-tumor-cell culture system supports the growth of malignant cells, that multiple cell populations exist in cell suspensions derived from solid tumors, and that differences exist between the radiosensitivity of cells at 2.0 Gy in different histology types

  2. Improvement In Rabbit Corneal Cell Suspension Viability After Freezing With Gingko Biloba Extrakt

    Directory of Open Access Journals (Sweden)

    Murad Aktan

    2007-01-01

    Full Text Available We investigated whether the addition of Gingko Biloba extract (EGb 761 to rabbit corneal epithelial medium before cell freezing improved cell viability after freezing then thawing. After removal of corneas, they were treated with enzymes and the corneal epithelium was prepared as a single cell suspension in freezing media with or without EGb 761. After freezing for two weeks then thawing, a higher cell viability was found in the cornea cell suspensions which had been frozen pretreated with EGb 761 in the media. The improvement with corneal cell viability with EGb 761 pretreatment is postulated to be based on the antioxidant capacity of the plant extract.

  3. Callogenesis and cell suspension establishment of tropical highland blackberry (Rubus adenotrichosSchltdl.) and its microscopic analysis.

    Science.gov (United States)

    Schmidt-Durán, Alexander; Alvarado-Ulloa, Carlos; Chacón-Cerdas, Randall; Alvarado-Marchena, Luis Fernando; Flores-Mora, Dora

    2016-01-01

    Blackberries are fruits produced worldwide, with 25 % of their production centered in Mexico, Central and South America. Tropical highland blackberry is a fruit that can potentially enhance human health, due to their high content in phenolic compounds, which include anthocyanins, phenolic acids, tannins (gallotannins and elagitannins) and flavonoids. Therefore, the overall aim of this study is the development of a callus induction protocol, the establishment of blackberry cell suspensions ( Rubus adenotrichos Schltdl.) and their cell analysis through optical microscopy and TEM, for the potential production of phenolic compounds. In order to produce callogenesis, segments of blackberry leaves were disinfected and placed in different concentrations of 2,4-D and the control media (0; 0.5; 1.0; 1.5; 2.0; 2.5 and 3.0 mg/l of 2,4-D); obtaining the higher size of calli in the medium with 1.5 mg/l of 2,4-D. After this determination, and for this specific treatment, a growth curve was performed through the use of fresh and dry weight parameters, in order to identify each of the growth stages. Furthermore, the calli obtained from the 1.5 mg/l of 2,4-D treatment were placed in two different culture media (MS and MS supplemented with 1.5 mg/l of 2,4-D) in order to establish the cell suspensions and the growth curve. To the best treatment, the total polyphenols were also quantified. It was determined that the MS medium is ideal for the growth and disintegration of the cell suspensions, obtaining 0.0256 mg of gallic acid/g of fresh sample. Finally, a cell callus and cell suspension analysis was performed through OM and TEM, evidencing a higher hystological differentiation in the calli, as well as the observation of antioxidant storage in the plastids.

  4. Histocytological analysis of callogenesis and somatic embryogenesis from cell suspensions of date palm (Phoenix dactylifera).

    Science.gov (United States)

    Sané, D; Aberlenc-Bertossi, F; Gassama-Dia, Y K; Sagna, M; Trouslot, M F; Duval, Y; Borgel, A

    2006-08-01

    The date palm is a dioecious perennial species of the Arecaceae for which in vitro micropropagation is essential to ensure the renewal of palm plantations. This study presents a histocytological analysis of the traditional Mauritanian Amsekhsi cultivar beginning from the initial callogenesis and continuing up to the establishment of the cellular embryogenic cell suspensions. The formation of somatic embryos and their development into rooted plants are also described. Foliar segments of seedlings cultured in the presence of 2,4-D produced primary calli that were chopped to produce fine friable granular calli that subsequently produced cellular suspensions when transferred to liquid medium. The somatic proembryos that developed after removal of the 2,4-D were plated on agar medium where they developed into rooted plants. Thin sections of tissue fragments taken at each stage of the process were stained using Periodic Acid Schiff and Naphthol Blue-Black. The first cellular divisions were localized close to the vascular vessels of the leaf. The primary calli were obtained within 2 months. Fine friable granular calli grew quickly after the primary calli were chopped. Individual embryogenic cells were identified that rapidly started to divide and developed into globular proembryos. In addition, in the microcalli, breaking zones appeared in the thick pectocellulosic walls which delimited the pluricellular proembryos. The anatomy of somatic embryos is similar to that of zygotic embryos despite a deficit in the accumulation of intracellular proteins. When rooted with NAA, the vitroplants developed a strong orthotropic taproot. This study contributes to understanding the whole process of somatic embryogenesis, but two specific questions remain to be answered: what factors are involved in the reactivation of the somatic cells at the beginning of the initial callogenesis, and why do the somatic embryos not accumulate proteins in their tissues during maturation?

  5. [Effect of auxins on production of coumarin in a suspension culture of Angelica archangelica L].

    Science.gov (United States)

    Siatka, T; Kasparová, M

    2003-07-01

    The paper examined the effect of selected auxins (2,4-dichlorophenoxyacetic acid, alpha-naphthalene-acetic acid, beta-indoleacetic acid, beta-indoleburytic acid; each in four concentrations--0.2, 2, 10, and 20 mg/l) on the production of coumarins in the suspension culture of Angelica archangelica L. cultinated in the dark and under permanent lighting(3500 lux). The effect of the light regimen is, in comparison with auxins, less marked--the content of coumarins is mostly comparable both under permanent lighting and in the dark. The highest coumarin content was achieved with the use of alpha-naphthalene-acetic acid in a concentration of 0.2 mg/l with cultivation in the dark.

  6. The development and evaluation of single cell suspension from wheat and barley as a model system; a first step towards functional genomics application.

    Science.gov (United States)

    Dong, Jing; Bowra, Steve; Vincze, Eva

    2010-11-05

    The overall research objective was to develop single cell plant cultures as a model system to facilitate functional genomics of monocots, in particular wheat and barley. The essential first step towards achieving the stated objective was the development of a robust, viable single cell suspension culture from both species. We established growth conditions to allow routine culturing of somatic cells in 24 well microtiter plate format. Evaluation of the wheat and barley cell suspension as model cell system is a multi step process. As an initial step in the evaluation procedure we chose to study the impact of selected abiotic stress elicitors at the physiological, biochemical and molecular level. We report the results of osmotic stress imposed by NaCl and PEG. As proline is an important osmoprotectant of the cereal cells, colorimetric assay for proline detection was developed for small volumes (200 μl). We performed RT-PCR experiments to study the change in the expression of the genes encoding Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Δ1-pyrroline-5-carboxylate reductase (PC5R) in response to abiotic stress. We found differences between the wheat and barley suspension cultures, barley being more tolerant to the applied osmotic stresses. We suggested a model to explain the obtained differences in stress tolerance between the two species. The suspension cell cultures have proven useful for determining changes in proline concentration and expression level of genes (P5CS, P5CR) under various treatments and we suggest that the cells can be used as a model host system to study gene expression and regulation in monocots.

  7. Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures.

    Science.gov (United States)

    Schöpke, C; Taylor, N; Cárcamo, R; Konan, N K; Marmey, P; Henshaw, G G; Beachy, R N; Fauquet, C

    1996-06-01

    A protocol was established for the introduction of DNA into embryogenic suspension-derived tissues of cassava via microparticle bombardment, for the selection of genetically transformed cells, and for the regeneration of fully transgenic plants from these cells. The plasmid DNA used for bombardment contained a gene encoding neomycin phosphotransferase (nptII) and a gene encoding beta-glucuronidase (uidA). Selection of bombarded tissue with paromomycin resulted in the establishment of putative transgenic embryogenic calli. In most of these calli, beta-glucuronidase was detected histochemically. Molecular analysis of paromomycin-resistant embryogenic calli and of plants regenerated from these calli, confirmed the stable integration of bombarded DNA into the cassava genome.

  8. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  9. Factors influencing cucumber (Cucumis sativus L. somatic embryogenesis. I. The crucial role of pH and nitrogen in suspension culture

    Directory of Open Access Journals (Sweden)

    Tadeusz Wróblewski

    2014-01-01

    Full Text Available A method of obtaining and the characteristics of an embryogenic stabilised cucumber (Cucumis sativus L. suspension culture which has many similarities to the carrot model are presented. The Specific Type I cells and proembryogenic mass were present in such a suspension. The maintenance of the proembryogenic stage took place in medium containing 2,4-D as the sole growth regulator, subsequent stages of embryogenesis occurred in hormone-free medium. Embryonic structures were also observed in medium with auxin in the late stages of growth, probably due to the depletion of 2,4-D in the medium during subculture. The choice of the proper inorganic nitrogen sources and the maintenance of correct proportions between them had a significant effect on the formation of these structures. We have shown that the pH of the medium with an embryogenic culture became stabilized regardless of the initial pH value and depended on the medium composition. The inoculum used for the initiation of subsequent subcultures of the stable suspension culture was 1 part tissue to 300 parts medium and was small in comparison to the systems described for the cucumber so far. From 1 ml of basic suspension 7 embryos were obtained on medium without growth regulators 10 days after inoculation, and this amount increased to 21 after 3 weeks. From 3.2% of the somatic embryos it was posible to regenerate plants. The high yield and synchronisation of the process and the development of embryos without passing through callus tissue create the possibility of using this system for molecular investigations and in the technology of somatic seed production.

  10. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    Science.gov (United States)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  12. In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

    Directory of Open Access Journals (Sweden)

    Cavalli Francesca

    2006-12-01

    Full Text Available Abstract Background Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established. Results Calli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used. Conclusion Here we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a

  13. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids.

    Science.gov (United States)

    Funk, C; Brodelius, P E

    1990-09-01

    Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-(14)C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia.

  14. CELL SHAPE AND HEXOSE TRANSPORT IN NORMAL AND VIRUS-TRANSFORMED CELLS IN CULTURE

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, M.J.; Farson, D.; Tung, A.S.C.

    1976-07-01

    The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer. The relation between the geometry of cells, transport rates, and growth regulation is undoubtedly very complex, and our knowledge of these relationships is still very elementary. In a recent review on the influence of geometry on control of cell growth, Folkman and Greenspan (1) pointed out that the permeability of cells in a flat versus a spherical state may indeed be very different. The growth properties of cells on a surface and in suspension have been compared often (1-5). However, with one exception. little is known about the changes in transport properties when cell shape is changed. Foster and Pardee (6) demonstrated that the active transport of a-aminoisobutyric acid was reduced 2.5 times in suspension cultures of Chinese hamster cells with respect to the cells grown on a coverslip. They attributed this to the smaller surface area of suspended cells. While it is not clear why active transport should be dependent on the surface area available, it is possible that once the cells assume a spherical configuration, the carrier proteins are redistributed in such a way as to make them less accessible to the substrate. What happens to

  15. ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

    International Nuclear Information System (INIS)

    David Gara, Pedro M.; Garabano, Natalia I.; Llansola Portoles, Manuel J.; Moreno, M. Sergio; Dodat, Diego; Casas, Oscar R.; Gonzalez, Mónica C.; Kotler, Mónica L.

    2012-01-01

    The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O 2 •− /HO 2 • , HO • , and H 2 O 2 generated upon 4-MeV X-ray irradiation of 6.4 μM silicon nanoparticle aqueous suspensions were on the order of 10 μM per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic 1 O 2 was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO 2 and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of 1 O 2 upon X-ray irradiation opens novel approaches in the design of therapy strategies.

  16. Computer Simulation Study of Collective Phenomena in Dense Suspensions of Red Blood Cells under Shear

    CERN Document Server

    Krüger, Timm

    2012-01-01

    The rheology of dense red blood cell suspensions is investigated via computer simulations based on the lattice Boltzmann, the immersed boundary, and the finite element methods. The red blood cells are treated as extended and deformable particles immersed in the ambient fluid. In the first part of the work, the numerical model and strategies for stress evaluation are discussed. In the second part, the behavior of the suspensions in simple shear flow is studied for different volume fractions, particle deformabilities, and shear rates. Shear thinning behavior is recovered. The existence of a shear-induced transition from a tumbling to a tank-treading motion is demonstrated. The transition can be parameterized by a single quantity, namely the effective capillary number. It is the ratio of the suspension stress and the characteristic particle membrane stress. At the transition point, a strong increase in the orientational order of the red blood cells and a significant decrease of the particle diffusivity are obser...

  17. Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions

    International Nuclear Information System (INIS)

    Begum, Parvin; Fugetsu, Bunshi

    2013-01-01

    Highlights: • This study was set up to explore potential influence of graphene on T87 cells. • Fragmented nuclei, membrane damage, mitochondrial dysfunction were observed. • ROS increased, ROS are key mediators in the cell death signaling pathway. • Translocation of graphene into cells and an endocytosis-like structure was observed. • Graphene entering into the cells by endocytosis. -- Abstract: The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0–80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2′,7′-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS

  18. Biostimulation effects of low-energy laser radiation on yeast cell suspensions

    Science.gov (United States)

    Anghel, Sorin; Stanescu, Constantin S.; Giosanu, Dana; Neagu, Ionica; Savulescu, Geta; Iorga-Siman, Ion

    2000-02-01

    This paper presents work to determine the effects produced by low energy laser radiation on the metabolism and growth of a yeast cell suspension. As experimental material, we used young yeast culture in liquid medium, then distributed on a solid medium, to obtain isolated colonies. As laser source, we used a He-Ne laser, and the irradiation was made with different exposure times. Form each irradiated material, a sample of white grape sterile must was sowed, that has fermented at 18 divided by 20 degrees C for 10 divided by 15 days, after that some properties was tested. Some microscopic studies were also made. The results prove some influence of low energy laser irradiation, which can induce mutations, with new properties of the irradiated material. These mutations can be obtained in a positive sense, with new and important perspectives in wine industry. Also, we observed an inhibitory effect of the laser radiation on the yeast cell growth, due, probably to the too high values of the exposure.

  19. Quinoline Alkaloids in Suspension Cultures of Cinchona ledgeriana Treated with Various Substances

    Directory of Open Access Journals (Sweden)

    DIAH RATNADEWI

    2010-12-01

    Full Text Available Cinchona alkaloids are in extensive uses, not only for drugs but also for soft drink industries. They are harvested from the bark of trees Cinchona spp. after certain ages and therefore are available over a limited time. Cell culture is an alternative way to continuously produce such secondary metabolites in a much shorter time. Various substances were added in the normal growth media to promote quinoline alkaloids production by cell cultures of Cinchona ledgeriana. At the sixth week of culture, quinine and cinchonine contents were suppressed by paclobutrazol (PBZ, abscisic acid (ABA, or even by precursor tryptophan, while cinchonidine content was enhanced by 0.2 mg/l tryptophan to 43 fold of that produced by untreated cells (2.8% dry weight. At the seventh week of culture, the production of quinine and quinidine started to grow whereas the production of cinchonine and cinchonidine tended to decrease. An addition of 5 mg/l PBZ to culture media yielded the highest level of total quinine/quinidine after seven weeks, e.g. quinine 11 times more abundant and quinidine 23 fold higher compared to the untreated cells. Particularly the level of quinine which is the most demanded for medical and industrial purposes still need to be improved to approach to or even higher than that of extracted from the conventional source.

  20. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  1. Cell culture compositions

    Science.gov (United States)

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

    2014-03-18

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

  2. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Jong, de A.J.; Yakimova, E.T.; Kapchina, V.M.; Woltering, E.J.

    2002-01-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and

  3. Optimizing cryopreservation of human spermatogonial stem cells: comparing the effectiveness of testicular tissue and single cell suspension cryopreservation.

    Science.gov (United States)

    Yango, Pamela; Altman, Eran; Smith, James F; Klatsky, Peter C; Tran, Nam D

    2014-11-01

    To determine whether optimal human spermatogonial stem cell (SSC) cryopreservation is best achieved with testicular tissue or single cell suspension cryopreservation. This study compares the effectiveness between these two approaches by using testicular SSEA-4+ cells, a known population containing SSCs. In vitro human testicular tissues. Academic research unit. Adult testicular tissues (n=4) collected from subjects with normal spermatogenesis and normal fetal testicular tissues (n=3). Testicular tissue versus single cell suspension cryopreservation. Cell viability, total cell recovery per milligram of tissue, as well as viable and SSEA-4+ cell recovery. Single cell suspension cryopreservation yielded higher recovery of SSEA-4+ cells enriched in adult SSCs, whereas fetal SSEA-4+ cell recovery was similar between testicular tissue and single cell suspension cryopreservation. Adult and fetal human SSEA-4+ populations exhibited differential sensitivity to cryopreservation based on whether they were cryopreserved in situ as testicular tissues or as single cells. Thus, optimal preservation of human SSCs depends on the patient's age, type of samples cryopreserved, and end points of therapeutic applications. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  4. Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus

    Directory of Open Access Journals (Sweden)

    Yazid Muhammad

    2010-10-01

    Full Text Available Abstract Background The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts. Results Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively. Conclusions In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.

  5. Poisson-Boltzmann theory of charged colloids: limits of the cell model for salty suspensions

    International Nuclear Information System (INIS)

    Denton, A R

    2010-01-01

    Thermodynamic properties of charge-stabilized colloidal suspensions and polyelectrolyte solutions are commonly modelled by implementing the mean-field Poisson-Boltzmann (PB) theory within a cell model. This approach models a bulk system by a single macroion, together with counterions and salt ions, confined to a symmetrically shaped, electroneutral cell. While easing numerical solution of the nonlinear PB equation, the cell model neglects microion-induced interactions and correlations between macroions, precluding modelling of macroion ordering phenomena. An alternative approach, which avoids the artificial constraints of cell geometry, exploits the mapping of a macroion-microion mixture onto a one-component model of pseudo-macroions governed by effective interparticle interactions. In practice, effective-interaction models are usually based on linear-screening approximations, which can accurately describe strong nonlinear screening only by incorporating an effective (renormalized) macroion charge. Combining charge renormalization and linearized PB theories, in both the cell model and an effective-interaction (cell-free) model, we compute osmotic pressures of highly charged colloids and monovalent microions, in Donnan equilibrium with a salt reservoir, over a range of concentrations. By comparing predictions with primitive model simulation data for salt-free suspensions, and with predictions from nonlinear PB theory for salty suspensions, we chart the limits of both the cell model and linear-screening approximations in modelling bulk thermodynamic properties. Up to moderately strong electrostatic couplings, the cell model proves accurate for predicting osmotic pressures of deionized (counterion-dominated) suspensions. With increasing salt concentration, however, the relative contribution of macroion interactions to the osmotic pressure grows, leading predictions from the cell and effective-interaction models to deviate. No evidence is found for a liquid

  6. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture

    Directory of Open Access Journals (Sweden)

    M Majumdar

    2014-01-01

    Full Text Available The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001, even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  7. Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2015-06-01

    Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

  8. Fluorescence emission spectra of calcofluor stained yeast cell suspensions: heuristic assessment of basis spectra for their linear unmixing.

    Science.gov (United States)

    Plášek, Jaromír; Dostál, Marek; Gášková, Dana

    2012-07-01

    Fluorescence emission spectra of yeast cell suspensions stained with calcofluor have recently been identified as promising markers of variations in the quality of yeast cell wall. It is shown in this paper how the raw fluorescence spectra of calcofluor can be transformed to reliable spectral signatures of cell wall quality, which are independent of actual dye-to-cell concentrations of examined cell suspensions. Moreover, the presented approach makes it possible to assess basis fluorescence spectra that allows for the spectral unmixing of raw fluorescence spectra in terms of respective fluorescence contributions of calcofluor solvated in the suspension medium and bound to yeast cell walls.

  9. Optimization of callus and cell suspension cultures of Barringtonia racemosa (Lecythidaceae family for lycopene production Otimização de culturas de suspensões de calos e células de Barringtonia racemosa (família Lecythidaceae para produção de licopeno

    Directory of Open Access Journals (Sweden)

    Mandana Behbahani

    2011-02-01

    Full Text Available Lycopene is present in a range of fresh fruits and vegetables, especially in the leaves of Barringtonia racemosa. The traditional lycopene extraction from the plant is being employed instead of an easy propagation technique like cell culture process from the leaf explants. We intend to assess how lycopene could be extracted via tissue culture under light (illuminance: 8,200 lux under white fluorescent lamps, photoperiod 16 h per day at 25ºC and dark. Leaf explants of Barringtonia racemosa were cultured on modified Murashige and Skoog (MS, Woody Plant Medium (WPM and B5 media, supplemented with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D. Optimal conditions for callus induction and maintenance under both dark and light were investigated, and growth and lycopene accumulation were evaluated. Among media with different concentrations of 2,4-D, fast growing, friable callus initiated within three weeks after culturing on WPM basal medium supplemented with 2.0 mg L-1 (weight per volume of 2,4-D, whereas callus induction in explants cultured on all other media started only after five weeks. Calli were subcultured once every fortnight. Pale yellow and green calli developed under conditions of dark and light respectively were then selected for evaluation of their lycopene contents. An improved reversed phase of high performance liquid chromatography (HPLC method was used for a selective chemical determination of the lycopene content. Light induced lycopene production; and likewise maximum lycopene level incubated in light was higher than those incubated in darkness. The best growth rates of callus and cell suspension were achieved in WPM and B5 media respectively. The production of lycopene was growth-dependent through analysis of growth and lycopene content of both callus and cell suspension cultures.O licopeno está presente numa série de frutas frescas e hortaliças principalmente na folhas de Barringtonia racemosa. A extra

  10. Ultrasound Characterization of Microbead and Cell Suspensions by Speed of Sound Measurements of Neutrally Buoyant Samples

    DEFF Research Database (Denmark)

    Cushing, Kevin W.; Garofalo, Fabio; Magnusson, Cecilia

    2017-01-01

    . The density of the microparticles is determined by using a neutrally buoyant selection process that involves centrifuging of microparticles suspended in different density solutions, CsCl for microbeads and Percoll for cells. The speed of sound at 3 MHz in the neutrally buoyant suspensions is measured...... and fixed cells, such as red blood cells, white blood cells, DU-145 prostate cancer cells, MCF-7 breast cancer cells, and LU-HNSCC-25 head and-neck squamous carcinoma cells in phosphate buffered saline. The results show agreement with published data obtained by other methods....

  11. Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation.

    Science.gov (United States)

    Fuentes, Alejandro; Ramos, Pedro Luis; Ayra, Camilo; Rodríguez, Meilyn; Ramírez, Nadia; Pujol, Merardo

    2004-06-01

    A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules. The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way. Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels. Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared. A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis. A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.

  12. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    Science.gov (United States)

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  13. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  14. Agrobacterium tumefaciens-mediated genetic transformation of embryogenesis cell suspensions of banana cultivar Grande naine (AAA

    Directory of Open Access Journals (Sweden)

    Idalmis Bermúdez-Caraballoso

    2004-01-01

    Full Text Available The black Sigatoka (Mycosphaerella fijiensis Morelet has become in the last years, the most destructive disease that affects the production of banana and plantains world-wide. The present work was made with the objective to obtain transgenic plants of banana cultivar Grand naine (AAA resistant to this disease with the use of genetic transformation. Embryogenenic cell suspensions obtained from somatic embryos formed from immature male flowers, were used for the transformation by Agrobacterium tumefaciens. The bacterial strain EHA-105 was used with the binary plasmids pHCA-58, pHCG-59 and pHGA-91, which contain different combinations of genes that encode for the antifungal chitinase, glucanase enzymes and the AP-24 osmotin. The commercial herbicide BASTA® was used as selective agent. One hundred ten putative transformed lines of the three constructions were obtained, after three selection months in the culture medium. The transgenic events were verified by means of Polymerase Chain Reaction analysis. Key words: AP-24, chitinase, glucanase, Musa, Mycosphaerella fijiensis

  15. Biotransformation of isonitrosoacetophenone (2-keto-2-phenyl-acetaldoxime) in tobacco cell suspensions

    CSIR Research Space (South Africa)

    Madala, NE

    2012-07-01

    Full Text Available Biotechnology Letters July 2012/ Vol. 34 No.7, pp 1351-1356 Biotransformation of isonitrosoacetophenone (2-keto-2- phenyl-acetaldoxime) in tobacco cell suspensions Ntakadzeni E. Madala 1 , P. A. Steenkamp 2 , L. A. Piater 1 and I. A. Dubery 1 1...

  16. Youth Culture and Cell Phone

    Directory of Open Access Journals (Sweden)

    mohammad saeed zokaei

    2009-11-01

    Full Text Available Iranian youth’s leisure culture has been immediately affected by the digital media culture. As a communicative media, cell phone has crossed borders of youth norms and identity; and in addition to facilitating their communication, has changed its patterns. Applying Bourdieu’s concepts of habitus and field, and relied on the qualitative and quantitative data gathered from the mobile youth users, the present study argues that mobile has produced a new field in which youth’s opportunities for leisure, entertainment, communication, and independence have extended. In addition, cell phone has facilitated and compensated for some defects in public sphere, and therefore empowered youth agency, individuality, and power. Despite this strengthening, cell phone does not cross borders of gender and class differences, or the levels of social capital.

  17. Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality.

    Science.gov (United States)

    Dill, Veronika; Hoffmann, Bernd; Zimmer, Aline; Beer, Martin; Eschbaumer, Michael

    2018-03-16

    Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A 24 -2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization

  18. Analysis of impedance measurements of a suspension of microcapsules using a variable length impedance measurement cell

    Directory of Open Access Journals (Sweden)

    Dejan Krizaj

    2012-10-01

    Full Text Available Electrical impedance measurements of the suspensions have to take into account the double layer impedance that is due to a very thin charged layer formed at the electrode-electrolite interface. A dedicated measuring cell that enables variation of the distance between the electrodes was developed for investigation of electrical properties of suspensions using two electrode impedance measurements. By varying the distance between the electrodes it is possible to separate the double layer and the suspension impedance from the measured data. From measured and extracted impedances electrical lumped models have been developed. The error of non inclusion of the double layer impedance has been analyzed. The error depends on the frequency of the measurements as well as on the distance between the electrodes.

  19. Cell thickness of UV absorption by the cell: relation to UV action spectrum shift in mammalian cells in culture

    International Nuclear Information System (INIS)

    Sakharov, V.H.; Voronkova, L.N.; Blokhin, A.V.

    1985-01-01

    By means of reconstruction of series half - thin transverse sections the three - dimensional morphometry of SPEV cells for a series of their specific states in culture is performed: for exponential growth in a monolayer, in a merged monolayer, in the mitosis phase, for giant cells and suspension cells. In the monolayer the cell thickness in its central part depended mainly on the nucleus thickness and in average changed but slightly despite a wide range of changes in volumes of nuclei and cells and their density in culture. The cell thickness has noticeably increased in mitosis. For the above states of cells UV radiation absorption spectra are determined. It is shown that a certain shift of action spectrus of death of mammalian cells as compared with that for bacterial cell can be a seguence of selfshielding and not differences in the nature of active chromophores

  20. In vitro plant regeneration from embryogenic cell suspension culture ...

    African Journals Online (AJOL)

    After two weeks, induction of somatic embryos up to the torpedo stage occured at all tested concentrations of 2,4-D, IAA or NAA. Somatic embryos developed only in MS medium containing 0.5 mg/l IAA within two weeks and 2% of globular embryos were developed into the cotyledonary stage embryos. Eighty one percent of ...

  1. Embryogenic callus formation, growth and regeneration in callus and suspension cultures of Miscanthus x ogiformis Honda 'Giganteus' as affected by proline

    DEFF Research Database (Denmark)

    Holme, Inger Bæksted; Krogstrup, Peter; Hansen, Jürgen

    1997-01-01

    to test the effect of proline in suspension cultures. The proline additions affected the formation of embryogenic callus and the growth of suspension cultures. Improvements depended on the proline concentration and the basal salts of the medium. Addition of 12.5 to 50 mM proline to callus induction medium...... with Murashige and Skoog salts increased embryogenic callus formation on shoot apices and leaf explants while proline had no effect on embryogenic callus formation in medium with N6 salts. Increased growth with increasing proline concentration was obtained in suspension aggregates grown in medium with N6 salts......The effects of proline additions to culture systems of Miscanthus x ogiformis Honda Giganteus' were investigated. Proline was added in concentrations of 0, 12.5, 25, 50, 100 or 300 mM to the callus induction and suspension culture media containing either Murashige and Skoog or N6 basal salts and 22...

  2. Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins.

    Science.gov (United States)

    Roowi, Siti Habsah; Ho, Chai-Ling; Alwee, Sharifah Shahrul Rabiah Syed; Abdullah, Meilina Ong; Napis, Suhaimi

    2010-09-01

    Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.

  3. Microtransplantation of dopaminergic cell suspensions: further characterization and optimization of grafting parameters.

    Science.gov (United States)

    Nikkhah, Guido; Rosenthal, Christoph; Falkenstein, Gero; Roedter, Alexandra; Papazoglou, Anna; Brandis, Almuth

    2009-01-01

    Intracerebral transplantation of dopaminergic (DA) cells is currently further explored as a potential restorative therapy for Parkinson's disease (PD). However, before they can be considered for a more widespread clinical use a number of critical issues have to be resolved, including an optimized transplantation protocol. This study has been performed in a rat 6-hydroxydopamine model of PD and is based on the microtransplantation approach. The results demonstrate a reduced survival (threefold) for a single cell suspension of E14 rat ventral mesencephalon compared to a fragment suspension when a metal cannula is used for implantation. However, fragment suspensions result in a more variable graft survival and ectopically placed cells along the implantation tract. When a glass capillary is used for implantation, the survival of the single cell suspension (so-called "micrograft") improved by fourfold (vs. single cells/metal cannula) and is superior to the combination of the metal cannula and fragment suspension (+40%). The micrografts show a reduced variability in DA neuron survival as well as fewer ectopically placed cells. Moreover, the implantation time can significantly be reduced from 19 to 7 min in micrografted animals without a compromise in DA graft survival and functional behavioral outcome. Using the microtransplantation approach graft size can be tailored effectively by varying the density of the final cell suspension at least between 11,000 and 320,000 cells/microl, resulting in comparable survival of tyrosine hydroxylase (TH)-positive neurons in the range of 2-4%. With this approach no more than 100 surviving TH-positive neurons are necessary to produce functional effects in the amphetamine-induced rotation test. Interestingly, we found that DA micrografts into lesion striatum present 20% higher survival rates of TH neurons in comparison to the intact striatum. In summary, these results provide further evidence for the usefulness of the

  4. Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

    International Nuclear Information System (INIS)

    Ecay, T.W.; Valentich, J.D.

    1991-01-01

    Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3 H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells

  5. NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

    International Nuclear Information System (INIS)

    Sullivan, S.G.; Rosenthal, J.S.; Winston, A.; Stern, A.

    1988-01-01

    NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red blood cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells. 9 refs.; 3 figs.; 1 table

  6. High-resolution 3-D imaging of living cells in suspension using confocal axial tomography.

    Science.gov (United States)

    Renaud, Olivier; Viña, Jose; Yu, Yong; Machu, Christophe; Trouvé, Alain; Van der Voort, Hans; Chalmond, Bernard; Shorte, Spencer L

    2008-01-01

    Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.

  7. A Highly Specific O-Methyltransferase for Nororientaline Synthesis Isolated from Argemone platyceras Cell Cultures.

    Science.gov (United States)

    Rueffer, M; Nagakura, N; Zenk, M H

    1983-12-01

    A highly specific new enzyme, S-adenosyl-L-methionine: (6-O-methyl-norlaudanosoline)-5'-O-methyltransferase which catalyses the formation of nor-orientaline from 6-O-methyl-norlaudanosoline and SAM was discovered, partially purified, and characterized. ARGEMONE PLATYCERAS cell suspension cultures served as enzyme source.

  8. Improved production of chlorogenic acid from cell suspension ...

    African Journals Online (AJOL)

    one third of Chinese traditional prescriptions [1,. 2]. ... The cultures were kept under continuous agitation at 110 rpm in an orbital shaker (Yiheng scientific instrument,. Shanghai,China) and incubated at 25 oC, with a .... Mean values with different letters are significantly different at p ≤ 0.05 according to Duncan's multiple.

  9. Characterization of technetium(vII) reduction by cell suspensions of thermophilic bacteria and archaea.

    Science.gov (United States)

    Chernyh, Nikolay A; Gavrilov, Sergei N; Sorokin, Vladimir V; German, Konstantin E; Sergeant, Claire; Simonoff, Monique; Robb, Frank; Slobodkin, Alexander I

    2007-08-01

    Washed cell suspensions of the anaerobic hyperthermophilic archaea Thermococcus pacificus and Thermoproteus uzoniensis and the anaerobic thermophilic gram-positive bacteria Thermoterrabacterium ferrireducens and Tepidibacter thalassicus reduced technetium [(99)Tc(VII)], supplied as soluble pertechnetate with molecular hydrogen as an electron donor, forming highly insoluble Tc(IV)-containing grayish-black precipitate. Apart from molecular hydrogen, T. ferrireducens reduced Tc(VII) with lactate, glycerol, and yeast extract as electron donors, and T. thalassicus reduced it with peptone. Scanning electron microscopy and X-ray microanalysis of cell suspensions of T. ferrireducens showed the presence of Tc-containing particles attached to the surfaces of non-lysed cells. This is the first report on the reduction in Tc(VII) by thermophilic microorganisms of the domain Bacteria and by archaea of the phylum Euryarchaeota.

  10. Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

    Science.gov (United States)

    Rakesh Minocha; Carolyn McQuattie; Wayne Fagerberg; Stephanie Long; Eun Woon Noh

    2001-01-01

    The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereo-logical and microscopic methods. Exposure to Al for 24-48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al....

  11. SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

    Science.gov (United States)

    Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

    2017-11-15

    Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell

  12. Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

    Science.gov (United States)

    Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

    2015-08-01

    Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  13. "Fungal elicitors combined with a sucrose feed significantly enhance triterpene production of a Salvia fruticosa cell suspension".

    Science.gov (United States)

    Kümmritz, Sibylle; Louis, Marilena; Haas, Christiane; Oehmichen, Franz; Gantz, Stephanie; Delenk, Hubertus; Steudler, Susanne; Bley, Thomas; Steingroewer, Juliane

    2016-08-01

    Oleanolic (OA) and ursolic acid (UA) are plant secondary metabolites with diverse pharmacological properties. To reach reasonable productivities with plant cell suspension cultures, elicitation is a widely used strategy. Within the presented work, the effects of different elicitors on growth and production of OA and UA in a Salvia fruticosa cell suspension culture were examined. Beside commonly used elicitors like jasmonic acid (JA) and yeast extract, the influence of medium filtrates of the endophytic fungi Aspergillus niger and Trichoderma virens was investigated. The best eliciting effects were achieved with JA and fungal medium filtrates. Both increased the triterpene content by approximately 70 %. Since JA showed significant growth inhibition, the volumetric triterpene yield did not increase. But, adding fungal filtrates increased the volumetric triterpene yield by approximately 70 % to 32.6 mgOA l(-1) and 65.9 mgUA l(-1) for T. virens compared to the control with 19.4 mgOA l(-1) and 33.3 mgUA l(-1). An elicitation strategy combining fungal medium filtrate of T. virens with sucrose feeding significantly enhanced cell dry weight concentration to 22.2 g l(-1) as well as triterpene content by approximately 140 %. In total, this led to an approximately 500 % increase of volumetric triterpene yield referring to the control with final values of 112.9 mgOA l(-1) and 210.4 mgUA l(-1). Despite the doubled cultivation duration, productivities of 6.7 mgOA l(-1) day(-1) and 12.4 mgUA l(-1) day(-1) were reached. These results demonstrate methods by which increased productivities of triterpenes can be achieved to attain yields competing with intact plants.

  14. ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

    Energy Technology Data Exchange (ETDEWEB)

    David Gara, Pedro M. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Garabano, Natalia I. [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina); Llansola Portoles, Manuel J. [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Moreno, M. Sergio [Centro Atomico Bariloche (Argentina); Dodat, Diego; Casas, Oscar R. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Gonzalez, Monica C., E-mail: gonzalez@inifta.unlp.edu.ar [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Kotler, Monica L., E-mail: kotler@qb.fcen.uba.ar [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina)

    2012-03-15

    The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O{sub 2}{sup Bullet -}/HO{sub 2}{sup Bullet}, HO{sup Bullet}, and H{sub 2}O{sub 2} generated upon 4-MeV X-ray irradiation of 6.4 {mu}M silicon nanoparticle aqueous suspensions were on the order of 10 {mu}M per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic {sup 1}O{sub 2} was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO{sub 2} and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of <5 nm size have the potential to be used as radiosensitizers for improving the outcomes of cancer radiotherapy. Their capability of producing {sup 1}O{sub 2} upon X-ray irradiation opens novel approaches in the design of therapy strategies.

  15. Preparation of labelled lipids by the use of plant cell cultures

    International Nuclear Information System (INIS)

    Mangold, H.K.

    1978-01-01

    The preparation of some radioacitvely labelled lipids by the use of plant cell cultures is discussed and further applications of the new method are suggested. Cell suspension cultures of rape (Brassica napus) and soya (Glycine max) have been used for the preparation of lipids labelled with radioisotopes. Radioactive acetic acid as well as various long-chain fatty acids are readily incorporated into the neutral and ionic lipids of plant cell cultures. In addition, 14 C-labelled glycerol, ethanolamine and choline are well utilized by the cells. Randomly labelled lipids have been obtained by incubating cell suspension cultures of rape and soya with [1- 14 C] acetic acid, and uniformly labelled lipids have been isolated from cultures that had been incubated with a mixture of [1- 14 C] acetic acid plus [2- 14 C] acetic acid. The use of techniques of plant cell cultures for the preparation of lipds labelled with stable or radioactive isotopesappears particularly rewarding because the uptake of precursors by the cells and their incorporation into various lipid compounds proceeds rapidly and often quanitatively.This new approach should be useful also for the biosynthesis of lipids whose acyl moieties contain a spn radical, a fluorescent group, or a light-sensitive label. Thus, plant cell cultures constitute valuable new tools for the biosynthetic preparation of a great variety of labelled lipids. (A.G.)

  16. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    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. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  17. Simple and inexpensive technique for measuring oxygen consumption rate in adherent cultured cells.

    Science.gov (United States)

    Takahashi, Eiji; Yamaoka, Yoshihisa

    2017-11-01

    Measurement of cellular oxygen consumption rate (OCR) is essential in assessing roles of mitochondria in physiology and pathophysiology. Classical techniques, in which polarographic oxygen electrode measures the extracellular oxygen concentration in a closed measuring vessel, require isolation and suspension of the cell. Because cell functions depend on the extracellular milieu including the extracellular matrix, isolation of cultured cells prior to the measurement may significantly affect the OCR. More recent techniques utilize optical methods in which oxygen-dependent quenching of fluorophores determines oxygen concentration in the medium at a few microns above the surface of the cultured cells. These techniques allow the OCR measurement in cultured cells adhered to the culture dish. However, this technique requires special equipment such as a fluorescence lifetime microplate reader or specialized integrated system, which are usually quite expensive. Here, we introduce a simple and inexpensive technique for measuring OCR in adherent cultured cells that utilizes conventional fluorescence microscopy and a glassware called a gap cover glass.

  18. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  19. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  20. Nanometer-scale sizing accuracy of particle suspensions on an unmodified cell phone using elastic light scattering.

    Directory of Open Access Journals (Sweden)

    Zachary J Smith

    Full Text Available We report on the construction of a Fourier plane imaging system attached to a cell phone. By illuminating particle suspensions with a collimated beam from an inexpensive diode laser, angularly resolved scattering patterns are imaged by the phone's camera. Analyzing these patterns with Mie theory results in predictions of size distributions of the particles in suspension. Despite using consumer grade electronics, we extracted size distributions of sphere suspensions with better than 20 nm accuracy in determining the mean size. We also show results from milk, yeast, and blood cells. Performing these measurements on a portable device presents opportunities for field-testing of food quality, process monitoring, and medical diagnosis.

  1. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

    Full Text Available Abstract Background In order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial. Objective By employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells. Methods Cigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors. Results Cigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

  2. Rotational magnetic pulses enhance the magnetofection efficiency in vitro in adherent and suspension cells

    Energy Technology Data Exchange (ETDEWEB)

    Dahmani, Ch., E-mail: dahmani@tum.de [Institute of Energy Conversion Technology, Technische Universität München, Munich (Germany); Mykhaylyk, O. [Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universität München, 81675 Munich (Germany); Helling, Fl. [Institute of Electrical Energy Supply, Universität der Bundeswehr, Munich (Germany); Götz, St. [Institute of Energy Conversion Technology, Technische Universität München, Munich (Germany); Weyh, Th. [Institute of Electrical Energy Supply, Universität der Bundeswehr, Munich (Germany); Herzog, H.-G. [Institute of Energy Conversion Technology, Technische Universität München, Munich (Germany); Plank, Ch. [Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar, Technische Universität München, 81675 Munich (Germany)

    2013-04-15

    The association of magnetic nanoparticles with gene delivery vectors in combination with the use of gradient magnetic fields (magnetofection) enables improved and synchronised gene delivery to cells. In this paper, we report a system comprising rotating permanent magnets to generate defined magnetic field pulses with frequencies from 2.66 to 133 Hz and a field amplitude of 190 or 310 mT at the location of the cells. Low-frequency pulses of 2.66–10 Hz with a magnetic flux density of 190 mT were applied to the examined cells for 30–120 s after magnetofection. These pulses resulted in a 1.5–1.9-fold enhancement in the transfection efficiency compared with magnetofection with only a static magnetic field in both adherent and suspension cells. The magnetic field amplitudes of 190 and 310 mT had similar effects on the transfection efficacy. No increase in the percentage of transgene-expressing suspension cells and no cytotoxic effects (based on the results of the MTT assay) were observed after applying alternating magnetic fields. - Highlights: ► We developed a magnetic system capable of generating defined magnetic pulses based on permanent magnets. ► The main advantage of the system is the lack of heat-induced fluctuations in the working parameters. ► Our system succeeded in enhancing the transfection of adherent human lung epithelial cells and human suspension cells. ► The enhancement in the transfection efficiency compared with static magnetic field is due to the magnetic field pulses. ► The approach could be used as a complementary method for drug targeting.

  3. Rotational magnetic pulses enhance the magnetofection efficiency in vitro in adherent and suspension cells

    International Nuclear Information System (INIS)

    Dahmani, Ch.; Mykhaylyk, O.; Helling, Fl.; Götz, St.; Weyh, Th.; Herzog, H.-G.; Plank, Ch.

    2013-01-01

    The association of magnetic nanoparticles with gene delivery vectors in combination with the use of gradient magnetic fields (magnetofection) enables improved and synchronised gene delivery to cells. In this paper, we report a system comprising rotating permanent magnets to generate defined magnetic field pulses with frequencies from 2.66 to 133 Hz and a field amplitude of 190 or 310 mT at the location of the cells. Low-frequency pulses of 2.66–10 Hz with a magnetic flux density of 190 mT were applied to the examined cells for 30–120 s after magnetofection. These pulses resulted in a 1.5–1.9-fold enhancement in the transfection efficiency compared with magnetofection with only a static magnetic field in both adherent and suspension cells. The magnetic field amplitudes of 190 and 310 mT had similar effects on the transfection efficacy. No increase in the percentage of transgene-expressing suspension cells and no cytotoxic effects (based on the results of the MTT assay) were observed after applying alternating magnetic fields. - Highlights: ► We developed a magnetic system capable of generating defined magnetic pulses based on permanent magnets. ► The main advantage of the system is the lack of heat-induced fluctuations in the working parameters. ► Our system succeeded in enhancing the transfection of adherent human lung epithelial cells and human suspension cells. ► The enhancement in the transfection efficiency compared with static magnetic field is due to the magnetic field pulses. ► The approach could be used as a complementary method for drug targeting

  4. A noninvasive technique for the measurement of the energetic state of free-suspension mammalian cells.

    Science.gov (United States)

    Ben-Tchavtchavadze, M; Chen, J; Perrier, Michel; Jolicoeur, Mario

    2010-01-01

    A perfusion small-scale bioreactor allowing on-line monitoring of the cell energetic state was developed for free-suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10-mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min(-1) for high-density cell suspensions (10(8) cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo (31)P NMR was performed and demonstrated the small-scale bioreactor platform ability to monitor the cell physiological behavior for 30-min experiments.

  5. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... used in conjunction with or in reference to cell cultures, which may be referred to as tissue cultures... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES...

  6. Altered nitrogen metabolism associated with de-differentiated suspension cultures derived from root cultures of Datura stramonium studied by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy.

    Science.gov (United States)

    Fliniaux, Ophélie; Mesnard, François; Raynaud-Le Grandic, Sophie; Baltora-Rosset, Sylvie; Bienaimé, Christophe; Robins, Richard J; Fliniaux, Marc-André

    2004-05-01

    De-differentiation of transformed root cultures of Datura stramonium has previously been shown to cause a loss of tropane alkaloid synthetic capacity. This indicates a marked shift in physiological status, notably in the flux of primary metabolites into tropane alkaloids. Nitrogen metabolism in transformed root cultures of D. stramonium (an alkaloid-producing system) and de-differentiated suspension cultures derived therefrom (a non-producing system) has been compared using Nuclear Magnetic Resonance (NMR) spectroscopy. (15)N-Labelled precursors [((15)NH(4))(2)SO(4) and K(15)NO(3)] were fed and their incorporation into nitrogenous metabolites studied using Heteronuclear Multiple Bond Coherence (HMBC) NMR spectroscopy. In both cultures, the same amino acids were resolved in the HMBC spectra. However, marked differences were found in the intensity of labelling of a range of nitrogenous compounds. In differentiated root cultures, cross-peaks corresponding to secondary metabolites, such as tropine, were observed, whereas these were absent in the de-differentiated cultures. By contrast, N- acetylputrescine and gamma-aminobutyric acid (GABA) accumulated in the de-differentiated cultures to a much larger extent than in the root cultures. It can therefore be suggested that the loss of alkaloid biosynthesis was compensated by the diversion of putrescine metabolism away from the tropane pathway and toward the synthesis of GABA via N-acetylputrescine.

  7. Increasing secondary metabolite production in plant-cell culture by redirecting transport.

    Science.gov (United States)

    Brodelius, P; Pedersen, H

    1993-01-01

    Various approaches for redirecting transport of secondary metabolites in plant-cell suspension cultures have been attempted in an effort to increase productivity. Little is understood of the transport mechanisms or their regulation, and many of the extractive methods which involve membrane permeabilization have an adverse effect on cell viability. Increasing the activity of metabolic pathways by elicitation, in conjunction with end-product removal and accumulation in an extractive phase, has proven to be the most successful strategy so far.

  8. Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

    Science.gov (United States)

    Ikeda, Kazuhiro; Nagata, Shogo; Okitsu, Teru; Takeuchi, Shoji

    2017-06-06

    Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

  9. Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel.

    Science.gov (United States)

    Hanna, Jacob; Hubel, Allison; Lemke, Erin

    2012-09-01

    Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 µm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations. Copyright © 2012 Wiley Periodicals, Inc.

  10. In vivo vascularization of cell sheets provided better long-term tissue survival than injection of cell suspension.

    Science.gov (United States)

    Takeuchi, Ryohei; Kuruma, Yosuke; Sekine, Hidekazu; Dobashi, Izumi; Yamato, Masayuki; Umezu, Mitsuo; Shimizu, Tatsuya; Okano, Teruo

    2016-08-01

    Cell sheets have shown a remarkable ability for repairing damaged myocardium in clinical and preclinical studies. Although they demonstrate a high degree of viability as engrafted cells in vivo, the reason behind their survivability is unclear. In this study, the survival and vascularization of rat cardiac cell sheets transplanted in the subcutaneous tissue of athymic rats were investigated temporally. The cell sheets showed significantly higher survival than cell suspensions for up to 12 months, using an in vivo bioluminescence imaging system to detect luciferase-positive transplanted cells. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay also showed a smaller number of apoptotic cells in the cell sheets than in the cell suspensions at 1 day. Rapid vascular formation and maturation were observed inside the cell sheets using an in vivo imaging system. Leaky vessels appeared at 6 h, red blood cells flowing through functional vessels appeared at 12 h, and morphologically matured vessels appeared at 7 days. In addition, immunostaining of cell sheets with nerve/glial antigen-2 (NG2) showed that vessel maturity increased over time. Interestingly, these results correlated with the dynamics of cell sheet mRNA expression. Genes related to endothelial cells (ECs) proliferation, migration and vessel sprouting were highly expressed within 1 day, and genes related to pericyte recruitment and vessel maturation were highly expressed at 3 days or later. This suggested that the cell sheets could secrete appropriate angiogenic factors in a timely way after transplantation, and this ability might be a key reason for their high survival. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  11. Modulated differential photoacoustic cell to study the gelatinization in a starch-water suspension

    Directory of Open Access Journals (Sweden)

    J. A. Villada

    2014-06-01

    Full Text Available In this paper the design and implementation of a novel Differential Photoacoustic Cell (DPC system is presented. The system was used to study the thermo optic transition in water-starch suspension called gelatinization. The melting temperature of Gallium was used to calibrate the temperature of the system. Both temperature values for starch gelatinization and gallium melting were agreed with those obtained using differential scanning calorimetry (DSC. The results show that this system is suitable to study other thermal processes in food or any thermal transition at low temperature.

  12. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  13. [Correction of cronic liver failure by transplantation of liver cells suspension and cell-engineering designs (experimental investigation)].

    Science.gov (United States)

    Got'e, S V; Shagidulin, M Iu; Onishchenko, N A; Krasheninnikov, M E; Il'inskiĭ, I M; Mozheĭko, N P; Liundup, A V; Volkova, E A; Petrakov, K I; Avramov, P V; Perova, N V; Sevast'ianov, V I

    2013-01-01

    On an experimental model of chronic fibrotic liver damage (male rats Wistar (n-60), damage of CCl4, the duration of the experiment 90 days) it was studied the effectiveness of cell therapy for the correction of chronic liver failure. These rats were divided into 3 experimental groups: in the Ist-group (control, n=10) isotonic saline (650 mkl.) was injected; in the IInd-group (n=20) suspension of liver cells was applicated in a dose 8 - l0 x 10(6) cells; in the IIIrd-group (n=30) suspension of liver cells and bone marrow cells (mesenchymal stromal cells) in ratio 5:1 were used as cell associates on microparticles intjectable heterogeneous biopolymer hydrogel "SpheroGEL" (cell-engineering design) in common dose 8 - l0 x 10(6) It was ascertained that in the 2nd and in the 3rd groups the accelerated normalization of disturbed liver functional indices (ALT, AST, ALP) took place - to 30 days, but in the control group only to 90 days. The reliable differences in rats ofnormalization offunctional indices were absent between the IInd and the IIIrd groups. But in 90 days by using special histological dyeing it was found out that defibrotic processes in liver tissue were more expressed in the IIIrd group in comparison with the IIIrd group. Received results were consequence of prolonged vital activity of cells (liver cells and mesenchymal stromal bone marrow cells) into cell-engineering designs, which were transplanted in the IIIrd group. The obtained effect can be explained by that the developed cell-engineering designs provide adequate conditions for prolonged vital activity of the transplanted cells.

  14. Analysis of redox relationships in the plant cell cycle: determinations of ascorbate, glutathione and poly (ADPribose)polymerase (PARP) in plant cell cultures.

    Science.gov (United States)

    Foyer, Christine H; Pellny, Till K; Locato, Vittoria; De Gara, Laura

    2008-01-01

    Reactive oxygen species (ROS) and low molecular weight antioxidants, such as glutathione and ascorbate, are powerful signaling molecules that participate in the control of plant growth and development, and modulate progression through the mitotic cell cycle. Enhanced reactive oxygen species accumulation or low levels of ascorbate or glutathione cause the cell cycle to arrest and halt progression especially through the G1 checkpoint. Plant cell suspension cultures have proved to be particularly useful tools for the study of cell cycle regulation. Here we provide effective and accurate methods for the measurement of changes in the cellular ascorbate and glutathione pools and the activities of related enzymes such poly (ADP-ribose) polymerase during mitosis and cell expansion, particularly in cell suspension cultures. These methods can be used in studies seeking to improve current understanding of the roles of redox controls on cell division and cell expansion.

  15. A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production

    Directory of Open Access Journals (Sweden)

    Tomomi G. Otsuji

    2014-05-01

    Full Text Available Utilizing human pluripotent stem cells (hPSCs in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

  16. Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

    Science.gov (United States)

    Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

    The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.

  17. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    Science.gov (United States)

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  18. Extracellular Synthesis of Luminescent CdS Quantum Dots Using Plant Cell Culture.

    Science.gov (United States)

    Borovaya, Mariya N; Burlaka, Olga M; Naumenko, Antonina P; Blume, Yaroslav B; Yemets, Alla I

    2016-12-01

    The present study describes a novel method for preparation of water-soluble CdS quantum dots, using bright yellow-2 (BY-2) cell suspension culture. Acting as a stabilizing and capping agent, the suspension cell culture mediates the formation of CdS nanoparticles. These semiconductor nanoparticles were determined by means of an UV-visible spectrophotometer, photoluminescence, high-resolution transmission electron microscopy (HRTEM), and XRD. Followed by the electron diffraction analysis of a selected area, transmission electron microscopy indicated the formation of spherical, crystalline CdS ranging in diameter from 3 to 7 nm and showed wurtzite CdS quantum dots. In the present work, the toxic effect of synthesized CdS quantum dots on Nicotiana tabacum protoplasts as a very sensitive model was under study. The results of this research revealed that biologically synthesized CdS nanoparticles in low concentrations did not induce any toxic effects.

  19. Use of plant cell cultures to study the metabolism of environmental chemicals

    International Nuclear Information System (INIS)

    Sandermann, H. Jr.; Scheel, D.; von der Trenck, T.

    1984-01-01

    The metabolism of the following environmental chemicals has been studied in cell suspension cultures of wheat (Triticum aestivum L.) and soybean (Glycine max L.):2, 4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), hexachlorobenzene, pentachlorophenol, diethylhexylphthalate , benzo [alpha] pyrene, and DDT. All chemicals tested, including the persistent ones, were partially metabolized. Polar conjugates predominated in all cases. A covalent incorporation into lignin could be demonstrated for 2,4-D and pentachlorophenol. A specific deposition in the cellular vacuole could be demonstrated for the beta-D-glucopyranoside conjugates derived from 2,4-D. A rapid assay procedure to evaluate the metabolism of a given 14 C-labeled chemical in plant cell suspension cultures is described. This procedure requires about 1 week, and the reproducibility of the results obtained has been assessed

  20. Bottlenecks in the generation and maintenance of morphogenic banana cell suspensions and plant regeneration via somatic embryogenesis therefrom

    Czech Academy of Sciences Publication Activity Database

    Schoofs, H.; Panis, B.; Strosse, H.; Mosqueda, A. M.; Torres, J. L.; Roux, N.; Doležel, Jaroslav; Swennen, R.

    2001-01-01

    Roč. 8, č. 2 (2001), s. 3-7 ISSN 0989-8972 R&D Projects: GA MŠk ME 376 Institutional research plan: CEZ:AV0Z5038910 Keywords : banana cell suspensions * plant regeneration Subject RIV: EA - Cell Biology

  1. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

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

  2. RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

    Directory of Open Access Journals (Sweden)

    Larsen Jeffrey S

    2012-05-01

    Full Text Available Abstract Background Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures. Results Replicating vectors derived from Potato virus X (PVX and Tobacco rattle virus (TRV were modified to contain the reporter gene β-glucuronidase (GUS with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass. Conclusions For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

  3. Isolation and culture of pulmonary endothelial cells.

    OpenAIRE

    Ryan, U S

    1984-01-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vas...

  4. Biophysical monitoring of cell cultures for quality assessment utilizing digital holographic microscopy

    Science.gov (United States)

    Kastl, Lena; Isbach, Michael; Dirksen, Dieter; Schnekenburger, Jürgen; Kemper, Björn

    2017-06-01

    Quality and reproducibility of cell-based assays strongly depend on the quality of the underlying cell culture which is influenced by various parameters like nutrient and growth factor availability, buffer conditions, subculture routines and optimal cell concentrations. Thus, methods for accurate assessment of objective cell parameters that characterize a specific cell line and detect global changes in cell culture are highly desirable. During the past years, quantitative phase imaging has been recognized as a promising tool for quantitative label-free live cell analysis. We demonstrate the utilization of quantitative phase imaging with digital holographic microscopy (DHM) to quantify the impact of cell culture conditions on single cells using a pancreatic tumor cell model. Label-free quantitative phase imaging of detached cells in suspension is performed by Michelson interferometer-based self-interference DHM. The quantitative phase images of the cells are analyzed for refractive index, volume and dry mass. We show that the evaluation of quantitative DHM phase images allows to extract absolute biophysical cellular parameters that are related to cell layer confluence states. In summary, the results of our study demonstrate that DHM is capable for label-free imaging cytometry with novel biophysical data sets that are acquired with minimum sample preparation for sophisticated monitoring of cell morphology alterations that are related to changes of cell culture conditions.

  5. First clues to understand red blood cell interactions: numerical studies of vesicle suspensions

    Science.gov (United States)

    Thiébaud, Marine; Misbah, Chaouqi; Dyfcom Team

    2014-03-01

    The scientific community started raising questions on blood flow for nearly two centuries, a period traced back to the pioneering work of Poiseuille. This topic has known a considerable upsurge of interest during the past decade. Vesicles capture several essential features shared with red blood cells. A single vesicle is now fairly understood, whereas study of suspensions is still unclear. We conduct bidimensionnal numerical studies by mean of the boundary integral method. Confinement plays a major role in that it introduces an interaction cut-off length. I will present results on the behavior of relative viscosity as function of the viscosity contrast between the fluid encapsulated by vesicles and the ambient fluid. This viscosity contrast is a key parameter: it triggers transition from tank-treading to tumbling regimes. Historical characterization of blood have led to the discovery of the Fahraeus-Lindqvist effect. I will introduce some results on this effect with a rheological study as function of concentration and confinement. I will report on non-standard behavior induced by a subtle spatio-temporal organization of the suspension. We would like to thank CNES for financial support.

  6. Optimizing the production of suspension cells using the G-Rex “M” series

    Directory of Open Access Journals (Sweden)

    Pradip Bajgain

    2014-01-01

    Full Text Available Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex “M” series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.

  7. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  8. [Glucose isomerase activity in suspension of Arthrobacter nicotianae cells and adsorption immobilization of the microorganisms on inorganic carriers].

    Science.gov (United States)

    Kovalenko, G A; Perminova, L V; Terent'eva, T G; Sapunova, L I; Lobanok, A G; Chuenko, T V; Rudina, N A; Cherniak, E I

    2008-01-01

    Kinetics of monosaccharide isomerization has been studied in suspensions of intact, non-growing Arthrobacter nicotianae cells. Under the conditions of the study, glucose and fructose were isomerized at the same maximum rate of 700 micromol/min per 1 g dried cells, which increased with temperature (the dependence was linear at 60-80 degrees C). The proposed means of adsorption immobilization of A. nicotianae cells involve inorganic carriers differing in macrostructure, chemical nature, and surface characteristics. Biocatalysts obtained by adsorbing the cells of A. nicotianae on carbon-containing foam ceramics in the coarse of submerged cultivation were relatively stable and retained original activity (catalysis of monosaccharide isomerization) throughout 14 h of use at 70 degrees C. Maximum glucose isomerase activity (2 micromol/min per 1 g) was observed with biocatalysts prepared by adsorption of non-growing A. nicotianae cells to the macroporous carbon-mineral carrier Sapropel and subsequent drying of the cell suspension together with the carrier.

  9. Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation.

    Science.gov (United States)

    Habaza, Mor; Kirschbaum, Michael; Guernth-Marschner, Christian; Dardikman, Gili; Barnea, Itay; Korenstein, Rafi; Duschl, Claus; Shaked, Natan T

    2017-02-01

    A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label-free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive-index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive-index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label-free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

  10. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

    Science.gov (United States)

    Onofre, J.; Baert, Y.; Faes, K.; Goossens, E.

    2016-01-01

    BACKGROUND Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23

  11. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation.

    Science.gov (United States)

    Onofre, J; Baert, Y; Faes, K; Goossens, E

    2016-11-01

    Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the

  12. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

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

    2007-01-01

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

  13. Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for Steviol glycoside production.

    Science.gov (United States)

    Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

    2014-03-01

    Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of NaCl (0.05-0.20%) and Na2CO3 (0.0125-0.10%) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57% with 0.10% NaCl, and 0.025% Na2CO3, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na2CO3 enhanced the SGs content from 1.36 (control) to 2.61 and 5.14%, respectively, on the 10th day.

  14. Isolation and culture of pulmonary endothelial cells.

    Science.gov (United States)

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.

  15. Metabolic cycles in primary metabolism of cell suspensions of Daucus carota L. analysed by C-NMR

    NARCIS (Netherlands)

    Krook, J.

    1999-01-01

    In the work described in this thesis, uptake and conversion of sugar by cells of batch-grown suspensions of Daucus carota L. were studied. Invasive techniques (measurements of enzyme activities and sugar and starch levels) and non-invasive techniques (

  16. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Seagrave, J.C.; Valdez, J.G.; Tobey, R.A.; Gurley, L.R.

    1985-06-01

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC1 2 , one being increased in amount while the other was reduced to an insignificant level. In ZnC1 2 -treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC1 2 -treated cells. While ZnC1 2 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC1 2 -treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC1 2 -treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  17. Henrietta Lacks, HeLa cells, and cell culture contamination.

    Science.gov (United States)

    Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

    2009-09-01

    Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.

  18. Electrical pulse – mediated enhanced delivery of silver nanoparticles into living suspension cells for surface enhanced Raman spectroscopy

    International Nuclear Information System (INIS)

    Lin, J; Li, B; Feng, S; Chen, G; Li, Y; Huang, Z; Chen, R; Yu, Y; Huang, H; Lin, S; Li, C; Su, Y; Zeng, H

    2012-01-01

    Electrical pulse-mediated enhanced silver nanoparticles delivery is a much better method for intracellular surface-enhanced Raman spectroscopy (SERS) measurements of suspension cells. Robust and high-quality SERS spectra of living suspension cells were obtained based on an electroporation-SERS method, which can overcomes the shortcoming of non-uniform distribution of silver nanoparticles localized in the cell cytoplasm after electroporation and reduces the amount variance of silver nanoparticles delivered into different cells. The electroporation parameters include three 150 V (375 V/cm) electric pulses of 1, 5, and 5 ms durations respectively. Our results indicate that considerable amount of silver nanoparticles can be rapidly delivered into the human promyelocytic leukemia HL60 cells, and the satisfied SERS spectra were obtained while the viability of the treated cells was highly maintained (91.7%). The electroporation-SERS method offers great potential approach in delivering silver nanoparticles into living suspension cells, which is useful for widely biomedical applications including the real-time intracellular SERS analysis of living cells

  19. Flow field measurements in the cell culture unit

    Science.gov (United States)

    Walker, Stephen; Wilder, Mike; Dimanlig, Arsenio; Jagger, Justin; Searby, Nancy

    2002-01-01

    The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the

  20. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

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

  1. Differentiation patterns of embryonic stem cells in two- versus three-dimensional culture.

    Science.gov (United States)

    Pineda, Emma T; Nerem, Robert M; Ahsan, Tabassum

    2013-01-01

    Pluripotent stem cells are attractive candidates as a cell source for regenerative medicine and tissue engineering therapies. Current methods of differentiation result in low yields and impure populations of target phenotypes, with attempts for improved efficiency often comparing protocols that vary multiple parameters. This basic science study focused on a single variable to understand the effects of two-dimensional (2D) versus three-dimensional (3D) culture on directed differentiation. We compared mouse embryonic stem cells (ESCs) differentiated on collagen type I-coated surfaces (SLIDEs), embedded in collagen type I gels (GELs), and in suspension as embryoid bodies (EBs). For a systematic analysis in these studies, key parameters were kept identical to allow for direct comparison across culture configurations. We determined that all three configurations supported differentiation of ESCs and that the kinetics of differentiation differed greatly for cells cultured in 2D versus 3D. SLIDE cultures induced overall differentiation more quickly than 3D configurations, with earlier expression of cytoskeletal and extracellular matrix proteins. For 3D culture as GELs or EBs, cells clustered similarly, formed complex structures and promoted differentiation towards cardiovascular phenotypes. GEL culture, however, also allowed for contraction of the collagen matrix. For differentiation towards fibroblasts and smooth muscle cells which actively remodel their environment, GEL culture may be particularly beneficial. Overall, this study determined the effects of dimensionality on differentiation and helps in the rational design of protocols to generate phenotypes needed for tissue engineering and regenerative medicine. Copyright © 2013 S. Karger AG, Basel.

  2. Methyl Jasmonate and Salicylic Acid Induced Oxidative Stress and Accumulation of Phenolics in Panax ginseng Bioreactor Root Suspension Cultures

    Directory of Open Access Journals (Sweden)

    Kee-Yoeup Paek

    2007-03-01

    Full Text Available To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 μM methyl jasmonate (MJ or salicylic acid (SA in a 5 L bioreactor suspension culture (working volume 4 L. Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2 contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH radical reducing activity were increased by MJ and SA. Fresh weight (FW and dry weight (DW decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%, DPPH activity (40%, flavonoids (88%, ascorbic acid (55%, NPSH (33%, and cysteine (62% contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and β-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

  3. New tissue dissociation protocol for scaled-up production of neural stem cells in suspension bioreactors.

    Science.gov (United States)

    Sen, Arindom; Kallos, Michael S; Behie, Leo A

    2004-01-01

    The successful dissociation of mammalian neural stem cell (NSC) aggregates (neurospheres) into a single-cell suspension is an important procedure when expanding NSCs for clinical use, or when performing important assays such as clonal analyses. Until now, researchers have had to rely primarily on destructive mechanical methods such as trituration with a pipette tip to break apart the aggregates. In this study we report on a new chemical dissociation procedure that is efficient, cost effective, reproducible, and much less harmful to murine NSCs than both mechanical and enzymatic techniques. This method, involving the manipulation of environmental pH levels, resulted in 40% higher measured cell densities and 15-20% higher viabilities compared with mechanical dissociation. Moreover, chemical dissociation resulted in the production of significantly less cellular debris. Chemical dissociation was found to have no adverse effects on the long-term proliferation of the NSCs, which retained the ability to proliferate, form neurospheres, self-renew, and exhibit multipotentiality. This chemical method represents a new approach for the dissociation of tissues.

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

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

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

  5. Biotransformation of thymol, carvacrol, and eugenol by cultured cells of Eucalyptus perriniana.

    Science.gov (United States)

    Shimoda, Kei; Kondo, Yoko; Nishida, Tomohisa; Hamada, Hatsuyuki; Nakajima, Nobuyoshi; Hamada, Hiroki

    2006-10-01

    The biotransformations of aroma compounds of spices, such as thymol (1), carvacrol (2), and eugenol (3), were investigated using cultured plant cells of Eucalyptus perriniana. Besides a beta-glucoside product (4, 3%), a biotransformation product, i.e., 5-methyl-2-(1-methylethyl)phenyl 6-O-(beta-d-glucopyranosyl)-beta-d-glucopyranoside (5, beta-gentiobioside, 87%), was isolated from the suspension cells after the five-day incubation of 1. On administration of 2, a beta-glucoside (6, 5%) and a beta-gentiobioside, i.e., 2-methyl-5-(1-methylethyl)phenyl 6-O-(beta-d-glucopyranosyl)-beta-d-glucopyranoside (7, 56%), were produced. Furthermore, E. perriniana cells also converted 3 into the corresponding beta-glucoside (8, 7%) and beta-gentiobioside (9, 58%). The cultured cells of E. perriniana are able to convert these aroma compounds of spices into glycosides which are accumulated in the cells.

  6. Proline and its metabolism enzymes in cucumber cell cultures during acclimation to salinity.

    Science.gov (United States)

    Naliwajski, Marcin R; Skłodowska, Maria

    2014-01-01

    Proline is an important osmolyte appearing as the result of salt stress response of plants. In the present study, we measured the proline concentration, activities of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and proline dehydrogenase (PDH) key regulatory enzymes in the biosynthesis and degradation of proline in the acclimated (AC20) and the non-acclimated (NAC) cucumber cell suspension cultures subjected to moderate (150 mM NaCl; AC20-150, NAC-150, respectively) and severe (200 mM NaCl; AC20-200, NAC-200, respectively) salt stress. The data showed that salt stress brought about a linear increase in proline content in both types of cultures. However, in the acclimated culture proline accumulation was observed earlier, in third hour after stress. Only in the acclimated culture moderate and severe stresses up-regulated P5CS activity throughout the experiment, whereas the activity of P5CR grew in response to both NaCl concentrations only in 24th and 48th hour. The severe salt stress resulted in decrease in P5CR in NAC-200 cultures. In response to salt stress, both types of cell suspension cultures reacted with decline in PDH activity below the spectrophotometrically detected level. Cell cultures vigor correlated with salt concentration and time of exposure to the stress factor. Both NaCl concentrations caused linear decline in vigor of the non-acclimated culture up to 80-90 % at the end of the experiment, whereas in the acclimated culture significant decrease by about 30-40 % was reached in 24th hour after stress. The presented data suggest that acclimation to salt stress up-regulated proline synthesis enzyme activity and caused intensive accumulations of proline by inhibiting its oxidation.

  7. Morphological changes in cultured bovine lymphoid cell lines associated with bovine viral diarrhea virus (BVDV) single and dual infections with bovine leukemia virus (BLV)

    Science.gov (United States)

    Currently, American Type Culture Collection (ATCC) makes available two cell lines derived from the same lymphoblast-like suspension cell that have been confirmed by next-generation sequencing and RT-PCR to have either a single contaminate of BVDV2a (CRL-8037) or dual contaminates of both BVDV and BL...

  8. Observation of antioxidant activity of leaves, callus and suspension ...

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... Antioxidant activity and phenolic compound was found in Justicia gendarussa via total phenolic content (TPC) and α,α-diphenyl-β-pycrilhydrazil hydrate (DPPH) radical scavenging assays. The assays were applied on aqueous and methanolic extracts of leaves, callus culture and cell suspension culture.

  9. Observation of antioxidant activity of leaves, callus and suspension ...

    African Journals Online (AJOL)

    Antioxidant activity and phenolic compound was found in Justicia gendarussa via total phenolic content (TPC) and ,-diphenyl--pycrilhydrazil hydrate (DPPH) radical scavenging assays. The assays were applied on aqueous and methanolic extracts of leaves, callus culture and cell suspension culture of J. gendarussa. Callus ...

  10. Effects of preculture with sucrose and aba on cell suspensions water status and its relation with vitrification resistance

    OpenAIRE

    SEIJO, GUILLERMO

    2000-01-01

    Changes in cell water relations during precultures were followed in an attempt to understand the mechanism of cell hardening for cryopreservation by vitrification. Medium containing 0.4 M sucrose (psiw=-1.45 MPa) and containing 5 mg L-1 of ABA (MT psiw=-0.73 MPa and MS psiw=-0.48 MPa) were used to harden cell suspensions of orange and carrot. Preculture in these medium did not cause a significant decrease of cell viability, however, it improved the cell survival to PVS2 and liquid nitrogen ex...

  11. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

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

  12. Cell Culture on MEMS Platforms: A Review

    Science.gov (United States)

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  13. [Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein].

    Science.gov (United States)

    Huang, Shigao; Yin, Yuting; Xiong, Chunhui; Wang, Caihong; Lü, Jianxin; Gao, Jimin

    2013-01-01

    In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

  14. Changes in Sexual Behavior of Orchidectomized Rats Under Influence of Allotransplantation of Testicular Interstitial Cell Suspension.

    Science.gov (United States)

    Deng, Bo; Bondarenko, Tatyana; Pakhomov, Oleksandr

    2017-05-09

    Transplantation of hormone-producing cells is an experimental endocrine dysfunction treatment. The present study investigated the effects of orchidectomy (OE) and transplantation of interstitial cell suspension (ICS) on rat sexual behavior. Adult experimental animals were divided into two populations. One of these populations had sexual experience before the experiment and the other did not. Each population was divided into three groups: control group and two orchidectomized groups. One of the orchidectomized groups was treated with ICS, and the other was treated with the vehicle. The changes in the sexual behavior were investigated on the following parameters: mount latency (ML), intromission latency (IL), ejaculation latency (EL), mount frequency (MF), intromission frequency (IF), copulatory efficacy (CE), and IF/EL ratio. The investigation of these changes lasted 4 weeks after ICS transplantation. The parameters of sexual behavior reflected a decrease in sexual function after OE at the beginning of the observation, especially for the animals that did not have a sexual experience. However, it was shown that sexual activity increased in the following 4 weeks. We have indicated that the loss of gonads attenuated the capacity to acquire sexual experience; nonetheless, it did not mean that the animals completely lost this capacity. Transplantation of ICS facilitated the maintenance of male sexual behavior after OE, fractionally enlarged the size of regressed seminal vesicles of the animals, and increased the free testosterone (T) level. These findings suggest the ICS can be considered as a temporal source of androgens, which can facilitate a restoration of sexual activity.

  15. Toxin- and cadmium-induced cell death events in tomato suspension cells resemble features of hypersensitive response

    NARCIS (Netherlands)

    Iakimova, E.T.; Woltering, E.J.; Yordanova, Z.P.

    2007-01-01

    Elicitors of different origin (fumonisin B1, fungal toxin), camptothecin (alkaloid from Camptotheca acuminata), mastoparan (wasp venom) and the heavy metal (cadmium) were tested for their ability to induce programmed cell death (PCD) in a model system of tomato cell culture, line MsK8. By employing

  16. Effects of radiation on cultured fish cells

    International Nuclear Information System (INIS)

    Etoh, Hisami; Suyama, Ippei

    1980-01-01

    A new fibroblastic cell line was established in our laboratory from the caudal fin of the goldfish, C. auratus. The cells, designated CAF, have been subcultured over 80 passages since initiation in August, 1977. A brief description of cell cultivation and colony formation is presented. The plating efficiency obtained was considerably higher than those reported for other fish cell lines. CAF cells were irradiated with 250, 500, 1,000, 2,000, and 3,000 R of x-rays at a dose rate of 80 R/min in air. The survival parameters changed when the number of passages of culture increased. Values for D 0 , D sub(q), and n obtained from cells irradiated at the 70th passage were calculated to be 650 R, 700 R, and 2.7 respectively. Thus CAF cells would be several times as resistant in general as cultured mammalian cells. The cells irradiated with 1,000 R of x-rays received a second dose from 250 to 2,000 R at intervals of 3, 6, and 24 hr. The cells kept at 26 0 C showed a pronounced recovery from sublethal damage during the intervals between two doses. Magnitude of recovery was larger if the interval was longer under the present experimental conditions. These results may indicate that the recovery observed at an individual level accounts partly for that in vitro. (author)

  17. Development, characterization and optimization of a new suspension chicken-induced pluripotent cell line for the production of Newcastle disease vaccine.

    Science.gov (United States)

    Shittu, Ismaila; Zhu, Ziying; Lu, Yangqing; Hutcheson, Jessica M; Stice, Steven L; West, Franklin D; Donadeu, Meritxell; Dungu, Baptiste; Fadly, Aly M; Zavala, Guillermo; Ferguson-Noel, Naola; Afonso, Claudio L

    2016-01-01

    Traditionally, substrates for production of viral poultry vaccines have been embryonated eggs or adherent primary cell cultures. The difficulties and cost involved in scaling up these substrates in cases of increased demand have been a limitation for vaccine production. Here, we assess the ability of a newly developed chicken-induced pluripotent cell line, BA3, to support replication and growth of Newcastle disease virus (NDV) LaSota vaccine strain. The characteristics and growth profile of the cells were also investigated. BA3 cells could grow in suspension in different media to a high density of up to 7.0 × 10(6) cells/mL and showed rapid proliferation with doubling time of 21 h. Upon infection, a high virus titer of 1.02 × 10(8) EID50/mL was obtained at 24 h post infection using a multiplicity of infection (MOI) of 5. In addition, the cell line was shown to be free of endogenous and exogenous Avian Leukosis viruses, Reticuloendotheliosis virus, Fowl Adenovirus, Marek's disease virus, and several Mycoplasma species. In conclusion, BA3 cell line is potentially an excellent candidate for vaccine production due to its highly desirable industrially friendly characteristics of growing to high cell density and capability of growth in serum free medium. Published by Elsevier Ltd.

  18. Cadmium toxicity in cultured tomato cells - Role of ethylene, proteases and oxidative stress in cell death signaling

    NARCIS (Netherlands)

    Iakimova, E.T.; Woltering, E.J.; Kapchina-Toteva, V.M.; Harren, F.J.M.; Cristescu, S.M.

    2008-01-01

    Our aim was to investigate the ability of cadmium to induce programmed cell death in tomato suspension cells and to determine the involvement of proteolysis, oxidative stress and ethylene. Tomato suspension cells were exposed to treatments with CdSO4 and cell death was calculated after fluorescein

  19. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

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

  20. The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F

    2000-01-01

    antibody Ber-EP4 was used as the primary capture antibody. In order to permit phenotyping of the isolated carcinoma cells the magnetic beads were removed from the carcinoma cells by DN'ase digestion of the DNA linker between the magnetic bead and the secondary antibody. In an ex vivo model system...... cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse...... an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found...

  1. Purification and characterization of cell suspensions peroxidase from cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Kouakou, Tanoh Hilaire; Dué, Edmond Ahipo; Kouadio, N'guessan Eugène Jean Parfait; Niamké, Sébastien; Kouadio, Yatty Justin; Mérillon, Jean-Michel

    2009-06-01

    Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 degrees C and was relatively stable at 20-30 degrees C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and beta-mercaptoethanol. Their activities were highly enhanced by Al(3+), Fe(3+), Ca(2+), and Ni(2+), but they were moderately inhibited by Mn(2+) and K(+). The enzyme lost 50% to 62% of its activity in the presence of Zn(2+) and Hg(2+).

  2. Growth and production of tropane alkaloids by cell suspension cutures of hyoscyamus albus L.

    OpenAIRE

    Kinsara, A. M. [احمد محمد كنسارة

    1990-01-01

    Cell suspension of Hyoscyamus albus L. were investigated for growth and production of tropane alkaloids in three different media. The effects of sucrose concentration and initial pH were also studied. 2,4- Dichlorophenoxyacetic acid and 1% sucrose enhanced the production of tropane alkaloids. The percent of alkaloids (0.75%) was higher than in the cultivated plant (0.352 %). أجرى قحص لنمو وانتاج قلويدات التروبان لمزارع المعلق الخلوي في ثلاث بيئات مختلفة لنبات السكران الأبيض ، وقد درس أيضا ...

  3. Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

    Science.gov (United States)

    Park, Eulsoon; Cho, Hong-Baek; Takimoto, Koichi

    2015-05-01

    Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Comparison of growth kinetics between static and dynamic cultures of human induced pluripotent stem cells.

    Science.gov (United States)

    Kato, Yuma; Kim, Mee-Hae; Kino-Oka, Masahiro

    2018-02-02

    Understanding the fundamental mechanisms that govern the growth kinetics of human induced pluripotent stem cells (hiPSCs) contributes to culture design strategies to improve large-scale production. Two hiPSC lines (Tic and 253G1) were cultured under static and dynamic suspension conditions, and growth kinetics were compared during early (24-48 h), middle (48-72 h), and late (72-96 h) stages. In 2D static culture, similar growth profiles were observed for both hiPSC lines. However, there were significant differences in growth profile patterns and aggregate morphologies between hiPSC lines grown in 3D static and dynamic cultures. Based on immunostaining comparing the two hiPSC lines, surface distribution of collagen type I was observed in aggregates of the Tic line, but not in those of the 253G1 line. Compared to that in 3D static culture, the numbers of cells at 96 h were significantly decreased in 3D dynamic culture. The apparent specific growth rate (μ app ) of the Tic line was maintained continuously throughout culture, whereas that of the 253G1 line decreased gradually with culture until the late phase, at which time this parameter was reduced to μ app  = (0.85 ± 0.71) × 10 -2  h -1 . This indicates that during the growth of hiPSCs in 3D dynamic culture, cells were damaged by liquid flow, which disrupted the cell-synthesized extracellular matrix (ECM). These results demonstrate that cell-synthesized ECM is an important factor affecting cell growth and morphology, and that changes to the ECM within aggregates lead to reduced growth abilities in dynamic culture. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-07-01

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

  6. Cellular heredity in haploid cultures of somatic cells. Progress report, August 1978-September 1979

    International Nuclear Information System (INIS)

    Freed, J.J.

    1979-10-01

    During the past year, studies were continued on the relation of ultraviolet mutagenesis to DNA repair in cultures of the haploid frog cell line ICR 2A. The method of irradiation of cells in suspension was improved by construction of an improved detector with major sensitivity to the 254 nm Hg resonance line, to give better estimates of actual exposure of the cells. Using this method, dose-response and dose-fractionation studies on irradiation of ouabain resistance were carried out. The uv induction of this phenotype in the ICR 2A cell line was found to be less than that necessary for adequate analysis of dose-response curves. Cell fusion experiments using frog and mouse cells revealed an enhancement of mutagenesis in the mouse parent that will be explored in further work

  7. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  8. Evaluation of treatment response to autologous transplantation of noncultured melanocyte/keratinocyte cell suspension in patients with stable vitiligo.

    Science.gov (United States)

    Ramos, Mariana Gontijo; Ramos, Daniel Gontijo; Ramos, Camila Gontijo

    2017-01-01

    Vitiligo is a chronic disease characterized by the appearance of achromic macules caused by melanocyte destruction. Surgical treatments with melanocyte transplantation can be used for stable vitiligo cases. To evaluate treatment response to the autologous transplantation of noncultured epidermal cell suspension in patients with stable vitiligo. Case series study in patients with stable vitiligo submitted to noncultured epidermal cell suspension transplantation and evaluated at least once, between 3 and 6 months after the procedure, to observe repigmentation and possible adverse effects. The maximum follow-up period for some patients was 24 months. Of the 20 patients who underwent 24 procedures, 25% showed an excellent rate of repigmentation, 50% good repigmentation, 15% regular, and 10% poor response. The best results were observed in face and neck lesions, while the worst in extremity lesions (88% and 33% of satisfactory responses, respectively). Patients with segmental vitiligo had a better response (84%) compared to non-segmental ones (63%). As side effects were observed hyperpigmentation of the treated area and the appearance of Koebner phenomenon in the donor area. Some limitations of the study included the small number of patients, a subjective evaluation, and the lack of long-term follow-up on the results. CONCLUSION: Noncultured epidermal cell suspension transplantation is efficient and well tolerated for stable vitiligo treatment, especially for segmental vitiligo on the face and neck.

  9. Serum Proteins Enhance Dispersion Stability and Influence the Cytotoxicity and Dosimetry of ZnO Nanoparticles in Suspension and Adherent Cancer Cell Models.

    Science.gov (United States)

    Anders, Catherine B; Chess, Jordan J; Wingett, Denise G; Punnoose, Alex

    2015-12-01

    Agglomeration and sedimentation of nanoparticles (NPs) within biological solutions is a major limitation in their use in many downstream applications. It has been proposed that serum proteins associate with the NP surface to form a protein corona that limits agglomeration and sedimentation. Here, we investigate the effect of fetal bovine serum (FBS) proteins on the dispersion stability, dosimetry, and NP-induced cytotoxicity of cationic zinc oxide nanoparticles (nZnO) synthesized via forced hydrolysis with a core size of 10 nm. Two different in vitro cell culture models, suspension and adherent, were evaluated by comparing a phosphate buffered saline (PBS) nZnO dispersion (nZnO/PBS) and an FBS-stabilized PBS nZnO dispersion (nZnO - FBS/PBS). Surface interactions of FBS on nZnO were analyzed via spectroscopic and optical techniques. Fourier transformed infrared spectroscopy (FTIR) confirmed the adsorption of negatively charged protein components on the cationic nZnO surface through the disappearance of surfaced-adsorbed carboxyl functional groups and the subsequent detection of vibrational modes associated with the protein backbone of FBS-associated proteins. Further confirmation of these interactions was noted in the isoelectric point shift of the nZnO from the characteristic pH of 9.5 to a pH of 6.1. In nZnO - FBS/PBS dispersions, the FBS reduced agglomeration and sedimentation behaviors to impart long-term improvements (>24 h) to the nZnO dispersion stability. Furthermore, mathematical dosimetry models indicate that nZnO - FBS/PBS dispersions had consistent NP deposition patterns over time unlike unstable nZnO/PBS dispersions. In suspension cell models, the stable nZnO - FBS/PBS dispersion resulted in a ~33 % increase in the NP-induced cytotoxicity for both Jurkat leukemic and Hut-78 lymphoma cancer cells. In contrast, the nZnO - FBS/PBS dispersion resulted in 49 and 71 % reductions in the cytotoxicity observed towards the adherent breast (T-47D) and prostate

  10. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  11. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

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

  12. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  13. Sensitivity of Toxorhynchites amboinensis mosquitoes versus chicken embryonic cell cultures for assays of Venezuelan encephalitis virus.

    Science.gov (United States)

    Scherer, W F; Chin, J

    1981-05-01

    Toxorhynchites amboinensis mosquitoes inoculated intrathoracically with Venezuelan encephalitis virus and tested for infectious virus 12 to 19 days later by inoculation of primary chicken embryonic cell cultures yielded approximately the same titers of virus as did direct inoculation of cultures, with counting of plaques 3 days thereafter. Titers were slightly higher in T. amboinensis for three virus strains, equal for two virus strains, and slightly lower for two virus strains. Comparative titers of four strains were similar, whether virus suspensions came from infectious vertebrate cells or infected invertebrate (T. amboinensis) cells. Each of the seven strains of Venezuelan encephalitis virus reached virus concentrations of 10(4.2) to 10(5.1) plaque-forming units in individual T. amboinensis mosquitoes after extrinsic incubation periods of 12 to 19 days. No temperature-sensitive (29 versus 37 degrees C) virus was detected in T. amboinensis mosquitoes infected by six strains of Venezuelan encephalitis virus.

  14. Role of cellular antioxidants (glutathione and ascorbic acid) in the growth and development of wild carrot suspension cultures

    International Nuclear Information System (INIS)

    Earnshaw, B.A.

    1986-01-01

    Determinations of endogenous glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid (AA) and dehydroascorbic acid (DHA) in proliferating and developing wild carrot cultures showed that lower levels of GSH and AA were associated with developing cultures. The GSSG and DHA levels did not account for the changes in the levels of antioxidants between proliferating and developing cultures. Studies were designed to test an observed auxin (2,4-Dichlorophenoxyacetic acid, 2,4-D)-antioxidant association. Two fractions (embryo and less developed) were obtained by screening developed cultures which were previously grown in the presence of 14 C-2, 4-D. The embryo fraction had a lower concentration of 14 C than the less developed fraction, supporting the association, since the two fractions showed this relationship with respect to GSH and AA concentrations. Determinations of GSH and AA levels of cells grown in various concentrations of 2,4-D showed the association, decreases in the 2,4-D concentration correlated with decreases in the GSH and AA concentrations. The existence of a respiratory pathway involving GSSG reductase, DHA reductase, and AA oxidase was investigated to test whether inhibition of AA oxidase by 2,4-D could explain the auxin-antioxidant association; however, AA oxidase activity was not detected

  15. [Effect of vanadium compounds on the growth and production of coumarins in the suspension culture of Angelica archangelica L].

    Science.gov (United States)

    Siatka, T; Kasparová, M

    2007-10-01

    Plant tissue cultures represent a promising source of substances of natural origin. The main problem of their use is a low production of the majority of secondary metabolites. One of the methods of increasing the production of these substances is elicitation, because the biosynthesis of many secondary metabolites in plant cells is part of the defensive reaction against biological or abiotic stress influences. The paper tested vanadium compounds as potential elicitors of the production of coumarins: sodium vanadate (0.2; 1; 10; 100 and 1000 microM/l of medium) and vanadyl sulfate (10; 20; 50; 100; 200 and 500 microM/l of medium). The toxicity of these substances for the culture was simultaneously monitored by means of the evaluation of the effects on growth (characterized by fresh and dry weights of biomass at the end of two-week cultivation). The cultures were grown both in the light and dark. The growth of the cultures was not influenced by sodium vanadate at concentrations of 0.2 to 100 microM. A vanadate concentration of 1000 microM acted already toxically (in comparison with the control culture, the fresh weight was decreased by 28 % and the dry weight by 41% when cultivated in the light; the fresh weight by 69% and the dry weight by 66% when cultivated in the dark). Vanadyl sulfate in concentrations of 10 to 50 microM did not affect the growth of the culture, at higher concentrations it decreased it gradually: a concentration of 500 microM acted already toxically, again more markedly when cultivated in the light (in comparison with the control culture, the fresh weight was decreased by 27% and dry weight by 38% when cultivated in the light; the fresh weight by 65% and the dry weight by 61% when cultivated in the dark). The production of coumarins was stimulated by sodium vanadate in a concentration of 0.2 and 1 microM when cultivated in the light. The content of coumarins increased in comparison with the control culture mainly in the medium by 46% and by 25

  16. Acetaldehyde and hexanaldehyde from cultured white cells

    Directory of Open Access Journals (Sweden)

    Zaldivar Frank

    2009-04-01

    Full Text Available Abstract Background Noninvasive detection of innate immune function such as the accumulation of neutrophils remains a challenge in many areas of clinical medicine. We hypothesized that granulocytes could generate volatile organic compounds. Methods To begin to test this, we developed a bioreactor and analytical GC-MS system to accurately identify and quantify gases in trace concentrations (parts per billion emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, frequently used to assess neutrophil function, was grown in serum-free medium. Results HL60 cells released acetaldehyde and hexanaldehyde in a time-dependent manner. The mean ± SD concentration of acetaldehyde in the headspace above the cultured cells following 4-, 24- and 48-h incubation was 157 ± 13 ppbv, 490 ± 99 ppbv, 698 ± 87 ppbv. For hexanaldehyde these values were 1 ± 0.3 ppbv, 8 ± 2 ppbv, and 11 ± 2 ppbv. In addition, our experimental system permitted us to identify confounding trace gas contaminants such as styrene. Conclusion This study demonstrates that human immune cells known to mimic the function of innate immune cells, like neutrophils, produce volatile gases that can be measured in vitro in trace amounts.

  17. Reproducible, ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.

    Directory of Open Access Journals (Sweden)

    Mark D Ungrin

    Full Text Available BACKGROUND: Human embryonic stem cells (hESC should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered. METHODOLOGY/PRINCIPAL FINDINGS: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2. CONCLUSIONS/SIGNIFICANCE: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.

  18. Effect of Agrobacterium rhizogenes and elicitation on the asiaticoside production in cell cultures of Centella asiatica

    Science.gov (United States)

    Ruslan, Komar; Selfitri, Anggrahaeni Dewi; Bulan, Shella A.; Rukayadi, Yaya; Elfahmi

    2012-01-01

    Background: Centella asiatica (L.) Urb. (Apiaceae) is an important medicinal plant, and it has been using to prepare herbal medicines. The compounds responsible for the biological activity of C. asiatica are triterpenoids such as asiaticoside. Asiaticoside is also important as a marker for standardization of C. asiatica. Due to the low content, there is a need to enhance the production of asiaticoside of C. asiatica. The biotechnological approach is one of the methods that can be used to enhance its production. Objectives: This study was designed to enhance the production of asiaticoside from C. asiatica using A. rhizogenes and elicitation experiments. Materials and Methods: Callus cultures were initiated using Murashige and Skoog (MS) medium supplemented with 1.0 mg/L indole-3-acetic acid (IAA) and 1.0 mg/L 6-benzylaminopurin (BAP). All media were supplemented with 4% (w/w) sucrose and solidified with 0.9% agar. Elicitations were done using pectin, methyl jasmonate, and Cu2+ ions. Transformed hairy root cultures were performed using A. rhizogenes. Results: Callus culture of C. asiatica was successfully initiated. Enhancement of the production of asiaticoside in the callus culture by elicitors pectin was up to 31%; methyl jasmonate (50 μM) in cell suspension cultures at day 14 was up to 171% compared to explant and 494% compared to control callus; copper ion (25 μM) at day 21 was up to 144% compared to explant, and 676% compared to control cell suspension cultures. While enhancement by genetic transformation using A. rhizogenes was 166-172% compare to untransformed roots Conclusion: Elicitation and genetically transformed hairy root cultures of C. asiatica produced asiaticoside up to 172% higher than untreated callus. PMID:22701283

  19. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

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

  20. Conversion of primordial germ cells to pluripotent stem cells: methods for cell tracking and culture conditions.

    Science.gov (United States)

    Nagamatsu, Go; Suda, Toshio

    2013-01-01

    Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

  1. Indirect immunofluorescence staining of cultured neural cells.

    Science.gov (United States)

    Barbierato, Massimo; Argentini, Carla; Skaper, Stephen D

    2012-01-01

    Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. This chapter describes procedures for the application of indirect immunofluorescence staining to neural cells in culture.

  2. Cultured epidermal stem cells in regenerative medicine.

    Science.gov (United States)

    Jackson, Catherine J; Tønseth, Kim Alexander; Utheim, Tor Paaske

    2017-07-04

    Transplantation of cultured epidermal cell sheets (CES) has long been used to treat patients with burns, chronic wounds, and stable vitiligo. In patients with large area burns this can be a life-saving procedure. The ultimate goal, however, is to restore all normal functions of the skin and prevent scar formation. Increased focus on the incorporation of epidermal stem cells (EpiSCs) within CES transplants may ultimately prove to be key to achieving this. Transplanted EpiSCs contribute to restoring the complete epidermis and provide long-term renewal.Maintenance of the regenerative potential of EpiSCs is anchorage-dependent. The extracellular matrix (ECM) provides physical cues that are interpreted by EpiSCs and reciprocal signaling between cells and ECM are integrated to determine cell fate. Thus, the carrier scaffold chosen for culture and transplant influences maintenance of EpiSC phenotype and may enhance or detract from regenerative healing following transfer.Long-term effectiveness and safety of genetically modified EpiSCs to correct the severe skin blistering disease epidermolysis bullosa has been shown clinically. Furthermore, skin is gaining interest as an easily accessible source of adult epithelial stem cells potentially useful for restoration of other types of epithelia. This review highlights the role of EpiSCs in the current treatment of skin injury and disease, as well as their potential in novel regenerative medicine applications involving other epithelia.

  3. Ascorbic acid transport into cultured pituitary cells

    International Nuclear Information System (INIS)

    Cullen, E.I.; May, V.; Eipper, R.A.

    1986-01-01

    An amidating enzyme designated peptidyl-glycine α-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 μM [ 14 C]ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 μM ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system

  4. Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts.

    Science.gov (United States)

    Kim, H-S; Son, H-Y; Kim, S; Lee, G-S; Park, C-H; Kang, S-K; Lee, B-C; Hwang, W-S; Lee, C-K

    2007-02-01

    The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.

  5. The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis.

    Science.gov (United States)

    Constabel, F; Shyluk, J P; Gamborg, O L

    1971-12-01

    Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10(-6)M 2,4-D. Transfer of cells to media with 10(-5)M kinetin or benzyladenine and no auxin or 10(-7)M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10(-5)M kinetin and with 10(-7) M NAA or 5×10(-5)M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10(-5)M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10(-7)M but not at 10(-6)M. At 10(-7)M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4-8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.

  6. Growth behavior in plant cell cultures based on emissions detected by a multisensor array.

    Science.gov (United States)

    Komaraiah, Palle; Navratil, Marian; Carlsson, Maria; Jeffers, Paul; Brodelius, Maria; Brodelius, Peter E; Kieran, Patricia M; Mandenius, Carl-Fredrik

    2004-01-01

    The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.

  7. Obtaining phenolic acids from cell cultures of various Artemisia ...

    African Journals Online (AJOL)

    Plant cell cultures represent a high valuable source for the production of bioactive secondary metabolites which can be used in food industry, medicine and cosmetic industry. In our study, we focused on obtaining phenolic acids from plant cell cultures. We compared cell cultures obtained from nine plant species of two ...

  8. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

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

    2004-01-01

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

  9. Molecular characterization of a cDNA encoding copper/zinc superoxide dismutase from cultured cells of Manihot esculenta.

    Science.gov (United States)

    Shin, Seung-Yong; Lee, Haeng-Soon; Kwon, Suk-Yoon; Kwon, Soon-Tae; Kwak, Sang-Soo

    2005-01-01

    Superoxide dismutase (SOD) cDNA, mSOD2, encoding cytosolic copper/zinc SOD (CuZnSOD) cDNA was isolated from suspension-cultured cells of cassava (Manihot esculenta Crantz) by cDNA library screening, and its expression was investigated in relation to environmental stress. mSOD2 is 774 bp in length with an open reading frame (ORF) of 152 amino acids, corresponding to a protein of predicted molecular mass 15 kDa and a pI of 5.22. One copy of the mSOD2 gene was found to be present in the cassava genome by Southern analysis using an mSOD2 cDNA-specific probe. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed diverse expression patterns for the mSOD2 gene in various tissues of intact cassava plants, at various stages of the growth in suspension cultures, and in the leaf tissues exposed to different stresses. The mSOD2 gene was highly expressed in suspension-cultured cells and in the stems of intact plants. However, it was expressed at low levels in leaves and roots. During suspension cell growth, the mSOD2 transcript progressively increased during culture. Moreover, the mSOD2 gene in excised cassava leaves responded to various stresses in different ways. In particular, it was highly induced in leaf tissue by several abiotic stresses, including high temperature (37 degrees C), chilling (4 degrees C), methyl viologen (MV) exposure, and wounding treatment. These results indicate that the mSOD2 gene is involved in the antioxidative process triggered by oxidative stress induced by environmental change.

  10. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  11. Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields

    International Nuclear Information System (INIS)

    Stevenson, A.P.; Tobey, R.A.

    1985-01-01

    Potassium ion influx was measured by monitoring 42 KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+

  12. Grafted fetal suprachiasmatic nucleus cells survive much better in tissue pieces than in suspension

    NARCIS (Netherlands)

    Boer, G. J.; Griffioen, H. A.; Saeed, P.

    1992-01-01

    A comparison was made between the survival of fetal suprachiasmatic nucleus (SCN) grafted either in tissue pieces or as tissue suspension. Donor tissue was obtained from day 15, 16 or 17 Wistar fetuses, and stereotaxically placed in the dorsal thalamus of the brain of vasopressin(VP)-deficient

  13. How variable is a spontaneous mutation rate in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Boesen, Jan J.B.; Niericker, Matthieu J.; Dieteren, Nicole; Simons, Jo W.I.M. (MGC-Dept. of Radiation Genetics and Chemical Mutagenesis, State Univ. of Leiden (Netherlands))

    1994-05-01

    The Luria-Delbrueck fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrueck fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2x10[sup -6] to about 2x10[sup -7]/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7[+-]4.0x10[sup -7] compared to 4.0[+-]3.1x10[sup -7] for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7[+-]1.3x10[sup -7]. Thus altogether the mutation rate can vary from 2x10[sup -6] to 0.7x10[sup -7] (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

  14. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...... compared to cell cultured in culture flasks incubated in a dark and CO2 conditioned incubator....

  15. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

    Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

    2017-12-01

    Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. An Integrated Bioprocess for the Expansion and Chondrogenic Priming of Human Periosteum-Derived Progenitor Cells in Suspension Bioreactors.

    Science.gov (United States)

    Gupta, Priyanka; Geris, Liesbet; Luyten, Frank P; Papantoniou, Ioannis

    2018-02-01

    The increasing use of microcarrier-based suspension bioreactors for scalable expansion of adult progenitor cells in recent years reveals the necessity of such approaches to address bio manufacturing challenges of advanced therapeutic medicinal products. However, the differentiation of progenitor cells within suspension bioreactors for the production of tissue modules is of equal importance but not well investigated. This study reports on the development of a bioreactor-based integrated process for expansion and chondrogenic priming of human periosteum-derived stem cells (hPDCs) using Cultispher S microcarriers. Spinner flask-based expansion and priming of hPDCs were carried out over 12 days for expansion and 14 days for priming. Characterization of the cells were carried out every 3rd day. Our study showed that hPDCs were able to expand till confluency with fold increase of 3.2±0.64 and to be subsequently primed toward a chondrogenic state within spinner flasks. During expansion, the cells maintained their phenotypic markers, trilineage differentiation capabilities and viability. Upon switching to TGF-β containing media the cells were able to differentiate toward chondrogenic lineage by clustering into mm-sized macrotissues containing hundreds of microcarriers. Chondrogenic priming was further evidenced by the expression of relevant markers at the mRNA level while maintaining their viability. Ectopic implantation of macrotissues highlighted that they were able to sustain their chondrogenic properties for 8 weeks in vivo. The method indicated here, suggests that expansion and relevant priming of progenitor cells can be carried out in an integrated bioprocess using spinner flasks and as such could be potentially extrapolated to other stem and progenitor cell populations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Bio-inactivation of human malignant cells through highly responsive diluted colloidal suspension of functionalized magnetic iron oxide nanoparticles

    Science.gov (United States)

    Ferreira, Roberta V.; Silva-Caldeira, Priscila P.; Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D.; Ardisson, José D.; Domingues, Rosana Z.

    2016-04-01

    Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions

  18. Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

    Directory of Open Access Journals (Sweden)

    Devaveena Dey

    Full Text Available BACKGROUND: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. METHODOLOGY: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. PRINCIPAL FINDINGS: We show that primary mammospheres contain a distinct side-population (SP that displays a CD24(low/CD44(low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high/CD24(low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1 mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. CONCLUSIONS: Thus, the self-renewal potential of human breast stem cells is

  19. Enhanced infectivity of bluetongue virus in cell culture by centrifugation.

    OpenAIRE

    Sundin, D R; Mecham, J O

    1989-01-01

    The effects of centrifugation of the infection of cell culture with bluetongue virus (BTV) were investigated. Baby hamster kidney cells were infected with BTV with or without centrifugation. Viral antigen was detected by immunofluorescence at 24 h in both centrifuged and noncentrifuged cultures. However, after 24 h of infection, the production of PFU in centrifuged cell cultures was 10- to 20-fold greater than that seen in cultures not centrifuged. In addition, centrifugation enhanced the dir...

  20. Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Nakano

    Full Text Available Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441 were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures, migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of

  1. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  2. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  3. Inhibition by derivatives of diguanidines of cell proliferation in Ehrlich ascites cells grown in cultures.

    Science.gov (United States)

    Alhonen-Hongisto, L; Pösö, H; Jänne, J

    1980-01-01

    The anti-proliferative effects of 1,1'-[(methylethanediylidene)dinitrilo]diguanidine [methylglyoxal bis(guanylhydrazone)] and 1,1'-[(metHYLETHANEDIYLIDENE)dinitrilo]bis-(3-aminoguaNIDINE) HAVE BEEN STUDIED IN Ehrlich ascites carcinoma cells grown in suspension cultures. Both compounds are potent inhibitors of S-adenosyl-L-methionine decarboxylase from the tumour cells. In the presence of putrescine (but not in its absence), the inhibition produced by 1,1'-[methylethanediylidene)dinitrilo]bis-(3-aminoguanadine) was apparently irreversible, as judged by persistent depression of the enzyme activity even after extensive dialysis. The two compounds produced similar increases in adenosylmethionine decarboxylase activity, which resulted from a striking stabilization of the enzyme in cells grown in the presence of the drugs. The inhibitory effect of the two diguanidine derivatives on the synthesis of DNA and protein became evident after an exposure of 4--8 h. At that time, the only change seen in tumour polyamines in cells grown in the presence of the inhibitors was an increase in cellular putrescine. To find out whether the compounds initially interfered with the energy production of the tumour cells, the cultures were grown in the presence of uniformly labelled glucose, and the formation of lactate, as well as the oxidation of the sugar into CO2, were measured. The activation of glycolysis upon dilution of the tumour cells with fresh medium and the subsequent formation of labelled CO2 were siliar in control cells and in cells exposed to methylglyoxal bis(buanylhydrazone), 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine) or diaminopropanol. Only a marginal decrease in the cellular content of ATP was found in cells exposed to the inhibitors for 24 h. The diguanidine-induced growth inhibition was fully reversed by low concentrations of exogenous polyamines. However, the possibility remained that the reversal by polyamines was due to a decrease of intracellular

  4. Differential astroglial responses in the spinal cord of rats submitted to a sciatic nerve double crush treated with local injection of cultured Schwann cell suspension or lesioned spinal cord extract: implications on cell therapy for nerve repair Respostas astrocitárias na medula espinal do rato submetido ao esmagamento duplo do nervo ciático e tratado com injeção local de suspensão de células de Schwann cultivadas ou de extrato de medula espinal lesada: implicações na terapia celular para o reparo do nervo

    Directory of Open Access Journals (Sweden)

    João Gabriel Martins Dallo

    2007-12-01

    Full Text Available PURPOSE: Reactive astrocytes are implicated in several mechanisms after central or peripheral nervous system lesion, including neuroprotection, neuronal sprouting, neurotransmission and neuropathic pain. Schwann cells (SC, a peripheral glia, also react after nerve lesion favoring wound/repair, fiber outgrowth and neuronal regeneration. We investigated herein whether cell therapy for repair of lesioned sciatic nerve may change the pattern of astroglial activation in the spinal cord ventral or dorsal horn of the rat. METHODS: Injections of a cultured SC suspension or a lesioned spinal cord homogenized extract were made in a reservoir promoted by a contiguous double crush of the rat sciatic nerve. Local injection of phosphate buffered saline (PBS served as control. One week later, rats were euthanized and spinal cord astrocytes were labeled by immunohistochemistry and quantified by means of quantitative image analysis. RESULTS: In the ipsilateral ventral horn, slight astroglial activations were seen after PBS or SC injections, however, a substantial activation was achieved after cord extract injection in the sciatic nerve reservoir. Moreover, SC suspension and cord extract injections were able to promote astroglial reaction in the spinal cord dorsal horn bilaterally. Conclusion: Spinal cord astrocytes react according to repair processes of axotomized nerve, which may influence the functional outcome. The event should be considered during the neurosurgery strategies.OBJETIVO: Astrócitos reativos participam de vários mecanismos após lesões do sistema nervoso central e periférico, os quais incluem neuroproteção, brotamento neuronal, neurotransmissão e dor neuropática. As células de Schwann (CS, um tipo de glia periférica, também reagem com a lesão do nervo, podendo interferir com o reparo e cicatrização, crescimento de fibras e regeneração neuronais. Investigamos aqui a possibilidade da terapia celular para o reparo do nervo ci

  5. Equipment for large-scale mammalian cell culture.

    Science.gov (United States)

    Ozturk, Sadettin S

    2014-01-01

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

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

    Science.gov (United States)

    Qin, Yanqin; Chen, Yulong; Li, Jiansheng

    2016-08-01

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

  7. Brownian Dynamics of a Suspension of Particles with Constrained Voronoi Cell Volumes

    KAUST Repository

    Singh, John P.

    2015-06-23

    © 2015 American Chemical Society. Solvent-free polymer-grafted nanoparticle fluids consist of inorganic core particles fluidized by polymers tethered to their surfaces. The attachment of the suspending fluid to the particle surface creates a strong penalty for local variations in the fluid volume surrounding the particles. As a model of such a suspension we perform Brownian dynamics of an equilibrium system consisting of hard spheres which experience a many-particle potential proportional to the variance of the Voronoi volumes surrounding each particle (E = α(Vi-V0)2). The coefficient of proportionality α can be varied such that pure hard sphere dynamics is recovered as α → 0, while an incompressible array of hairy particles is obtained as α →. As α is increased the distribution of Voronoi volumes becomes narrower, the mean coordination number of the particle increases and the variance in the number of nearest neighbors decreases. The nearest neighbor peaks in the pair distribution function are suppressed and shifted to larger radial separations as the constraint acts to maintain relatively uniform interstitial regions. The structure factor of the model suspension satisfies S(k=0) → 0 as α → in accordance with expectation for a single component (particle plus tethered fluid) incompressible system. The tracer diffusivity of the particles is reduced by the volume constraint and goes to zero at φ 0.52, indicating an earlier glass transition than has been observed in hard sphere suspensions. The total pressure of the suspension grows in proportion to (αkBT)1/2 as the strength of the volume-constraint potential grows. This stress arises primarily from the interparticle potential forces, while the hard-sphere collisional contribution to the stress is suppressed by the volume constraint.

  8. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-06-01

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

  9. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

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

    1997-01-01

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

  10. Efficient definitive endoderm induction from mouse embryonic stem cell adherent cultures: A rapid screening model for differentiation studies

    Directory of Open Access Journals (Sweden)

    Josué Kunjom Mfopou

    2014-01-01

    Full Text Available Definitive endoderm (DE differentiation from mouse embryonic stem cell (mESC monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGFβ and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies.

  11. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    Science.gov (United States)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  12. Radiosensitivity of hematopoietic stem cells in diffusion chamber cultures of the murine yolk sac and adult medullary tissue

    International Nuclear Information System (INIS)

    Weinberg, S.R.; Stohlman, F. Jr.

    1976-01-01

    The radiosensitivity of hematopoietic progenitor cells derived from the 10 1 / 2 day murine yolk sac and adult murine bone marrow have been compared. A yolk sac cell suspension was exposed to varying amounts of gamma radiation (0 to 950 R) and the index used for radiosensitivity was the proliferation and differentiation of these cells cultured for 10 days in the millipore diffusion chamber. The data provided evidence that the hematopoietically active embryonic yolk sac is less radiosensitive than the adult mouse tibial marrow cells. A 10 1 / 2 day yolk sac cell suspension (5.182 +- 0.601 x 10 6 cells per yolk sac) contained at least 2.8 +- 1.1 pluripotential stem cells (colony-forming units, CFU) and 242 +- 31 committed stem cells (colony-forming cells, CFC). The results suggest the existence of a hematopoietic stem cell compartment in the yolk sac which is markedly different from that found in adult medullary tissue. Furthermore, there is a difference between the expressions of the differentiation potential of precursor cells of extraembryonic and of adult origin when exposed to similar microenvironments

  13. Thermo-radiosensitivity of the granulocyte and macrophage precursor cells of mice. I.-Development of the in vivo culture and effects induced by the hyperthermia

    International Nuclear Information System (INIS)

    Bueren, J. A.; Nieto, M.

    1983-01-01

    The present report shows the agar diffusion chamber technique for culturing granulocyte- macrophage precursor cells, obtained from mice bone marrow. Diffusion chambers containing the bone marrow suspension are implanted intraperitoneally Into mice and constitute a compartment which avoids the migration of cells, but allows the transit of the mouse biological fluxes, necessary for the cellular proliferation. By means of this technique, we studied the lethal effects of the hyperthermia on the precursors and their capacity to repair sublethal damage. (Author) 129 refs

  14. Load-cell based characterization system for a "Violin-Mode" shadow-sensor in advanced LIGO suspensions.

    Science.gov (United States)

    Lockerbie, N A; Tokmakov, K V

    2016-07-01

    The background to this work was a prototype shadow sensor, which was designed for retro-fitting to an advanced LIGO (Laser Interferometer Gravitational wave Observatory) test-mass/mirror suspension, in which 40 kg test-mass/mirrors are each suspended by four approximately 600 mm long by 0.4 mm diameter fused-silica suspension fibres. The shadow sensor comprised a LED source of Near InfraRed (NIR) radiation and a rectangular silicon photodiode detector, which, together, were to bracket the fibre under test. The aim was to detect transverse Violin-Mode resonances in the suspension fibres. Part of the testing procedure involved tensioning a silica fibre sample and translating it transversely through the illuminating NIR beam, so as to measure the DC responsivity of the detection system to fibre displacement. However, an equally important part of the procedure, reported here, was to keep the fibre under test stationary within the beam, whilst trying to detect low-level AC Violin-Mode resonances excited on the fibre, in order to confirm the primary function of the sensor. Therefore, a tensioning system, incorporating a load-cell readout, was built into the test fibre's holder. The fibre then was excited by a signal generator, audio power amplifier, and distant loudspeaker, and clear resonances were detected. A theory for the expected fundamental resonant frequency as a function of fibre tension was developed and is reported here, and this theory was found to match closely with the detected resonant frequencies as they varied with tension. Consequently, the resonances seen were identified as being proper Violin-Mode fundamental resonances of the fibre, and the operation of the Violin-Mode detection system was validated.

  15. Load-cell based characterization system for a “Violin-Mode” shadow-sensor in advanced LIGO suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Lockerbie, N. A.; Tokmakov, K. V. [SUPA (Scottish Universities Physics Alliance) Department of Physics, University of Strathclyde, 107 Rottenrow, Glasgow G4 0NG (United Kingdom)

    2016-07-15

    The background to this work was a prototype shadow sensor, which was designed for retro-fitting to an advanced LIGO (Laser Interferometer Gravitational wave Observatory) test-mass/mirror suspension, in which 40 kg test-mass/mirrors are each suspended by four approximately 600 mm long by 0.4 mm diameter fused-silica suspension fibres. The shadow sensor comprised a LED source of Near InfraRed (NIR) radiation and a rectangular silicon photodiode detector, which, together, were to bracket the fibre under test. The aim was to detect transverse Violin-Mode resonances in the suspension fibres. Part of the testing procedure involved tensioning a silica fibre sample and translating it transversely through the illuminating NIR beam, so as to measure the DC responsivity of the detection system to fibre displacement. However, an equally important part of the procedure, reported here, was to keep the fibre under test stationary within the beam, whilst trying to detect low-level AC Violin-Mode resonances excited on the fibre, in order to confirm the primary function of the sensor. Therefore, a tensioning system, incorporating a load-cell readout, was built into the test fibre’s holder. The fibre then was excited by a signal generator, audio power amplifier, and distant loudspeaker, and clear resonances were detected. A theory for the expected fundamental resonant frequency as a function of fibre tension was developed and is reported here, and this theory was found to match closely with the detected resonant frequencies as they varied with tension. Consequently, the resonances seen were identified as being proper Violin-Mode fundamental resonances of the fibre, and the operation of the Violin-Mode detection system was validated.

  16. Load-cell based characterization system for a "Violin-Mode" shadow-sensor in advanced LIGO suspensions

    Science.gov (United States)

    Lockerbie, N. A.; Tokmakov, K. V.

    2016-07-01

    The background to this work was a prototype shadow sensor, which was designed for retro-fitting to an advanced LIGO (Laser Interferometer Gravitational wave Observatory) test-mass/mirror suspension, in which 40 kg test-mass/mirrors are each suspended by four approximately 600 mm long by 0.4 mm diameter fused-silica suspension fibres. The shadow sensor comprised a LED source of Near InfraRed (NIR) radiation and a rectangular silicon photodiode detector, which, together, were to bracket the fibre under test. The aim was to detect transverse Violin-Mode resonances in the suspension fibres. Part of the testing procedure involved tensioning a silica fibre sample and translating it transversely through the illuminating NIR beam, so as to measure the DC responsivity of the detection system to fibre displacement. However, an equally important part of the procedure, reported here, was to keep the fibre under test stationary within the beam, whilst trying to detect low-level AC Violin-Mode resonances excited on the fibre, in order to confirm the primary function of the sensor. Therefore, a tensioning system, incorporating a load-cell readout, was built into the test fibre's holder. The fibre then was excited by a signal generator, audio power amplifier, and distant loudspeaker, and clear resonances were detected. A theory for the expected fundamental resonant frequency as a function of fibre tension was developed and is reported here, and this theory was found to match closely with the detected resonant frequencies as they varied with tension. Consequently, the resonances seen were identified as being proper Violin-Mode fundamental resonances of the fibre, and the operation of the Violin-Mode detection system was validated.

  17. Load-cell based characterization system for a “Violin-Mode” shadow-sensor in advanced LIGO suspensions

    International Nuclear Information System (INIS)

    Lockerbie, N. A.; Tokmakov, K. V.

    2016-01-01

    The background to this work was a prototype shadow sensor, which was designed for retro-fitting to an advanced LIGO (Laser Interferometer Gravitational wave Observatory) test-mass/mirror suspension, in which 40 kg test-mass/mirrors are each suspended by four approximately 600 mm long by 0.4 mm diameter fused-silica suspension fibres. The shadow sensor comprised a LED source of Near InfraRed (NIR) radiation and a rectangular silicon photodiode detector, which, together, were to bracket the fibre under test. The aim was to detect transverse Violin-Mode resonances in the suspension fibres. Part of the testing procedure involved tensioning a silica fibre sample and translating it transversely through the illuminating NIR beam, so as to measure the DC responsivity of the detection system to fibre displacement. However, an equally important part of the procedure, reported here, was to keep the fibre under test stationary within the beam, whilst trying to detect low-level AC Violin-Mode resonances excited on the fibre, in order to confirm the primary function of the sensor. Therefore, a tensioning system, incorporating a load-cell readout, was built into the test fibre’s holder. The fibre then was excited by a signal generator, audio power amplifier, and distant loudspeaker, and clear resonances were detected. A theory for the expected fundamental resonant frequency as a function of fibre tension was developed and is reported here, and this theory was found to match closely with the detected resonant frequencies as they varied with tension. Consequently, the resonances seen were identified as being proper Violin-Mode fundamental resonances of the fibre, and the operation of the Violin-Mode detection system was validated.

  18. Cholera toxin stimulation of human mammary epithelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  19. Yeast suspension filtration: Flux enhancement using an upward gas/liquid slug flow -- Application to continuous alcoholic fermentation with cell recycle

    Energy Technology Data Exchange (ETDEWEB)

    Mercier, M.; Maranges, C.; Fonade, C.; Lafforgue-Delorme, C. [Institut National des Sciences Appliquees, Toulouse (France). Centre de Bioingenierie Gilbert Durand

    1998-04-05

    This study deals with the use of an upward gas/liquid slug flow to reduce tubular mineral membrane fouling. The injection of air into the feedstream is designed to create hydrodynamic conditions that destabilize the cake layer over the membrane surface inside the filtration module complex. Experimental study was carried out by filtering a biological suspension (yeast) through different tubular mineral membranes. The effects of operating parameters, including the nature of the membrane, liquid and gas flowrates, and transmembrane pressure, were examined. When external fouling was the main limiting phenomenon, flux enhancements of a factor of three could be achieved was gas sparging compared with single liquid phase crossflow filtration. The economic benefits of this unsteady technique have also been examined. To investigate the possibility of long-term operation of the two-phase flow principle, dense cell perfusion cultures of Saccharomyces cerevisiae were carried out in a fermentor coupled with an ultrafiltration module. The air injection allowed a high and stable flux to be maintained over 100 h of fermentation, with a final cell concentration of 150 g dry weight/L. At equal biomass level, a twofold gain in flux could be attained compared with classical steady crossflow filtration at half the cost.

  20. Electrospinning of microbial polyester for cell culture

    International Nuclear Information System (INIS)

    Kwon, Oh Hyeong; Lee, Ik Sang; Ko, Young-Gwang; Meng, Wan; Jung, Kyung-Hye; Kang, Inn-Kyu; Ito, Yoshihiro

    2007-01-01

    Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture

  1. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    of the microfluidic perfusion cell culture system is shown by investigation of adipose-derived stem cell (ASC) differentiation into adipocytes, where we have revealed that paracrine/autocrine signaling is involved in differentiation of a population of ASCs into adipocytes. We have thereby demonstrated......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs...

  2. Can established cultured papilloma cells harbor bovine papillomavirus?

    Science.gov (United States)

    Campos, S R C; Trindade, C; Ferraz, O P; Giovanni, D N S; Lima, A A; Caetano, H V A; Carvalho, R F; Birgel, E H; Dagli, M L Z; Mori, E; Brandão, P E; Richtzenhain, L J; Beçak, W; Stocco, R C

    2008-10-21

    Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

  3. Metabolism of monoterpenes in cell cultures of common sage (Salvia officinalis)

    International Nuclear Information System (INIS)

    Falk, K.L.; Gershenzon, J.; Croteau, R.

    1990-01-01

    Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes ( 14 C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1,000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism

  4. Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium

    International Nuclear Information System (INIS)

    Baker, F.L.; Spitzer, G.; Ajani, J.A.

    1986-01-01

    The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves

  5. [Effects on proliferation ability of vascular smooth muscle cells by static and/or dynamic cell culture: utility of pre-seeding technique for dynamic cell culture].

    Science.gov (United States)

    Yokomuro, Hiroki; Ozawa, Tsukasa; Fujii, Takeshiro; Shiono, Noritsugu; Watanabe, Yoshinori; Yoshihara, Katsunori; Koyama, Nobuya; Okada, Mitsumasa

    2007-11-01

    Conventional biomaterials are not viable, do not grow, and do not provide contractile effects in cardiac tissue. Foreign synthetic material may become thrombogenic or infected. The most recent cardiac constructs consist of biodegradable material which has the potential to solve these problems. However, dynamic three-dimensional cell culture is necessary because conventional culture is limited to construct tough biografts. Vascular smooth muscle cells derived from rat aorta were seeded to poly-L-lactide-epsilon-capro-lactone copolymer in three groups; static culture group (static cell seeding + static cell culture), dynamic culture group (dynamic cell seeding + dynamic cell culture), and pre-seeding group [static cell seeding and culture for 1 week (pre-seeding) + dynamic cell culture]. The dynamic cell culture system used an original spinner flask. The pre-seeding technique used static cell seeding and culture before dynamic culture. The three groups were evaluated by cell proliferation and histologic studies. Vascular smooth muscle cells could be proliferated in/on the biodegradable materials. The pre-seeding group cells grew much more efficiently than the other groups. Very few cells were found in the biodegradable materials with the dynamic groups. However, there were many cells in the materials with the static culture group and pre-seeding group, especially the pre-seeding group. Dynamic culture is useful for constructing tough biografts by the pre-seeding technique.

  6. Stem Cell Heterogeneity of Mononucleated Cells from Murine Peripheral Blood: Molecular Analysis

    Directory of Open Access Journals (Sweden)

    Muhammad Dain Yazid

    2011-01-01

    Full Text Available The main purpose of this paper was to determine the heterogeneity of primary isolated mononucleated cells that originated from the peripheral blood system by observing molecular markers. The isolated cells were cultured in complete medium for 4 to 7 days prior to the separation of different cell types, that is, adherent and suspension. Following a total culture time of 14 days, adherent cells activated the Cd105 gene while suspension cells activated the Sca-1 gene. Both progenitor markers, Cbfa-1 and Ostf-1, were inactivated in both suspension and adherent cells after 14-day culture compared to cells cultured 3 days in designated differentiation medium. In conclusion, molecular analyses showed that primary mononucleated cells are heterogeneous, consisting of hematopoietic stem cells (suspension and mesenchymal stem cells (adherent while both cells contained no progenitor cells.

  7. Experimental infection of New Zealand Merino sheep with a suspension of Mycobacterium avium subspecies paratuberculosis (Map) strain Telford: Kinetics of the immune response, histopathology and Map culture.

    Science.gov (United States)

    Dukkipati, Venkata S R; Ridler, Anne L; Thompson, Keith G; Buddle, Bryce M; Hedgespeth, Barry A; Price-Carter, Marian; Begg, Douglas J; Whittington, Richard J; Gicquel, Brigitte; Murray, Alan

    2016-11-15

    A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johne's disease in NZ Merino lambs. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

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

  9. Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

    Science.gov (United States)

    Hanga, Mariana P.; Heathman, Thomas R. J.; Coopman, Karen; Nienow, Alvin W.; Williams, David J.; Hewitt, Christopher J.

    2017-01-01

    ABSTRACT Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination

  10. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-04

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

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

    Science.gov (United States)

    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)

  12. Synthesis of polymer materials for use as cell culture substrates

    Energy Technology Data Exchange (ETDEWEB)

    Lakard, Sophie [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, IUT, 30 Avenue de l' Observatoire, 25009 Besancon (France)], E-mail: sophie.lakard@univ-fcomte.fr; Morrand-Villeneuve, Nadege [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Lesniewska, Eric [Laboratoire de Physique de l' Universite de Bourgogne, University of Bourgogne, 9 Avenue Savary, 21078 Dijon (France); Lakard, Boris [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Michel, Germaine [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Herlem, Guillaume [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Gharbi, Tijani [Laboratoire d' Optique P.M. Duffieux, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Fahys, Bernard [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France)

    2007-12-20

    Up to today, several techniques have been used to maintain cells in culture for studying many aspects of cell biology and physiology. More often, cell culture is dependent on proper anchorage of cells to the growth surface. Thus, poly-L-lysine, fibronectin or laminin are the most commonly used substrates. In this study, electrosynthesized biocompatible polymer films are proposed as an alternative to these standard substrates. The electrosynthesized polymers tested were polyethylenimine, polypropylenimine and polypyrrole. Then, the adhesion, proliferation and morphology of rat neuronal cell lines were investigated on these polymer substrates in an attempt to develop new and efficient polymer materials for cell culture. During their growth on the polymers, the evolution of the cell morphology was monitored using both confocal microscopy and immunohistochemistry, leading to the conclusion of a normal development. An estimation of the adhesion and proliferation rates of rat neuronal cell cultures indicated that polyethylenimine and polypropylenimine were the best substrates for culturing olfactory neuronal cells. A method to favour the differentiation of the neuronal cells was also developed since the final aim of this work is to develop a biosensor for odour detection using differentiated neuronal cells as transducers. Consequently, a biosensor was microfabricated using silicon technology. This microsystem allowed us to culture the cells on a silicon wafer and to position the cells on certain parts of the silicon wafer.

  13. Development of a microfluidic perfusion 3D cell culture system

    Science.gov (United States)

    Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

    2018-04-01

    Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

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

    Science.gov (United States)

    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.

  15. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  16. Preparation, characterisation and antibacterial activity of a florfenicol-loaded solid lipid nanoparticle suspension.

    Science.gov (United States)

    Wang, Ting; Chen, Xiaojin; Lu, Mengmeng; Li, Xihe; Zhou, WenZhong

    2015-12-01

    A florfenicol-loaded solid lipid nanoparticle (FFC-SLN) suspension was prepared by hot homogenisation and ultrasonic technique. The suspension was characterised for its release profile, stability, toxicity, and the physicochemical properties of the nanoparticles. Antibacterial activity of the suspension was evaluated in vitro and in vivo. The results showed that the mean diameter, polydispersity index and zeta potential of the nanoparticles were 253 ± 3 nm, 0.409 ± 0.022 and 47.5 ± 0.21 mV, respectively. In vitro release profile showed the FFC-SLN suspension had sustained release effect. The minimum inhibition concentration values of the FFC-SLN suspension were 6 and 3 µg/mL against Staphylococcus aureus and Escherichia coli respectively, compared with 3.5 and 2 µg/mL of native florfenicol. The suspension was relatively stable at 4°C and less stable at room temperature during 9 months storage. Although the nanoparticle carriers exhibited cytotoxicity in cell cultures, the LD50 of the lyophilised dry power of the suspension was higher than 5 g/kg body weight. Mortality protection against E. coli lethal infection in mice showed that the nanoparticle suspension had much better efficacy (6/10) than native drug (1/10). These results indicate that FFC-SLN suspension could be a promising formulation in veterinary medicine.

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

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

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

  18. [Studies on the lymphatic tropism and lymph cells apoptosis of cisplatin-nano carbon suspension in the rats].

    Science.gov (United States)

    Hou, Minmin; Chen, Zhuo; Li, Li; Wang, Ping; Qie, Mingrong

    2014-11-01

    To research the lymphatic tropism and lymph cell apoptosis of cisplatin-nano carbon suspension in rats with the aim of proposing a new way for chemotherapy. A total of 72 Wistar rats were randomly divided into two groups. For the experimental group, cisplatin-nano carbon suspension 0.3 ml (4 mg/ml) was injected subcutaneously into Wistar rats' plantar. For the control group, cisplatin 0.3 ml (4 mg/ml) was injected intravenously. Cisplatin concentration in the inguinal lymphatic tissue and plasma was determined by high performance liquid chromatography (HPLC) at 1, 2, 3, 4, 5 and 6 hours after drug administration. The apoptosis of lymph cell was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay (TUNEL). Targeting ability were evaluated and compared by targeting index (TI), selecting index (SI) and relative extraction efficiency (RE). SPSS 17.0 statistical software was used to analyze the differentiation of the cisplatin concentration and apoptosis index (AI) among various groups. DAS software was used to evaluate the lymphatic tropism. The cisplatin concentration of lymphatic tissue in experimental group were respectively (1.03± 0.32), (3.00±0.91), (2.20±0.73), (1.56±0.38), (1.30±0.74) and (0.78±0.34)µg/g after administration 1, 2, 3, 4, 5 and 6 hours, while in control group were (0.49±0.21), (1.02±0.70), (0.59±0.50), (0.56±0.21), (0.47±0.18) and (0.36±0.13)µg/g, in which there were significant difference at every times (all P tropism, and could send cisplatin to lymphatic tissue to achieve a higher concentration. The trait may break a new way for chemotherapy targeting lymph metastasis.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  20. Induction of interdigitating cell processes in podocyte culture.

    Science.gov (United States)

    Yaoita, Eishin; Yoshida, Yutaka; Nameta, Masaaki; Takimoto, Hiroki; Fujinaka, Hidehiko

    2018-02-01

    Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  1. Viable Cell Culture Banking for Biodiversity Characterization and Conservation.

    Science.gov (United States)

    Ryder, Oliver A; Onuma, Manabu

    2018-02-15

    Because living cells can be saved for indefinite periods, unprecedented opportunities for characterizing, cataloging, and conserving biological diversity have emerged as advanced cellular and genetic technologies portend new options for preventing species extinction. Crucial to realizing the potential impacts of stem cells and assisted reproductive technologies on biodiversity conservation is the cryobanking of viable cell cultures from diverse species, especially those identified as vulnerable to extinction in the near future. The advent of in vitro cell culture and cryobanking is reviewed here in the context of biodiversity collections of viable cell cultures that represent the progress and limitations of current efforts. The prospects for incorporating collections of frozen viable cell cultures into efforts to characterize the genetic changes that have produced the diversity of species on Earth and contribute to new initiatives in conservation argue strongly for a global network of facilities for establishing and cryobanking collections of viable cells.

  2. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    Science.gov (United States)

    Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Trinh, Tinh T. (Inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  3. The release of iron by Sertoli cells in culture

    NARCIS (Netherlands)

    Wauben-Penris, P. J.; Veldscholte, J.; van der Ende, A.; van der Donk, H. A.

    1988-01-01

    In seminiferous tubules, iron transport from the blood to the abluminal germinal cells must occur through the Sertoli cell cytoplasm. We investigated the release of previously accumulated iron by cultured Sertoli cells. We found that Sertoli cells contain easily releasable and less easily releasable

  4. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

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

  5. Studies on binding of radiolabeled thyrotropin to cultured human thyroid cells

    International Nuclear Information System (INIS)

    Yamamoto, M.; Rapoport, B.

    1978-01-01

    A line of cultured human thyroid adenoma cells was used in a study designed to compare the stimulatory effect of TSH on cellular cAMP generation with the binding of radiolabeled TSH to the cells. At 37 C, specific binding of [ 125 I]TSH to suspensions of thyroid cells was maximal at 20 min and was reversed by the addition of excess TSH. Unlike the generation of cellular cAMP in response to TSH stimulation, which was maximal at pH 7.5, the binding of [ 125 ]TSH to the cells was maximal at pH 5.5 and progressively declined up to pH 8.5. Increasing NaCl concentrations progressively inhibited cellular binding of TSH; at physiological salt concentrations, almost no TSH binding was detectable. Competitive inhibition studies of [ 125 I]TSH binding to cells revealed a binding site with a dissociation constant of 5.5 x 10 -8 M at pH 7.4. GH, PRL, hCG, FSH, insulin, and glucagon did not compete with [ 125 I)TSH binding. ACTH, however, was a potent inhibitor of [ 125 I]TSH binding. Despite this inhibitory effect on TSH binding, ACTH had little or no effect on cellular cAMP generation. High concentrations of ACTH did not inhibit the biological effect of TSH on cAMP generation. Specific binding of [ 125 I]TSH to empty plastic culture dishes was time dependent, reversible, and displayed a hormonal specificity identical to binding to thyroid cells. The effects of pH and NaCl concentrations on TSH binding to dishes were similarbut not identical to those on cellular binding. This study raises serious questions as to the biological significance of [ 125 I]TSH binding to cultured human thyroid cells

  6. Culture of porcine luteal cells as a substrate for in vitro maturation of porcine cumulus oocyte complexes. Establishment and characterization

    Directory of Open Access Journals (Sweden)

    Teplitz MA

    2016-12-01

    Full Text Available The aim of this study was to establish and characterize the porcine luteal cells (PLC culture for the subsequent coculture with porcine COC. The final purpose is to promote the oocyte maturation. The PLC was established using corpora lutea obtained from slaughterhouse ovaries. Corpora lutea were dissected and luteal tissue submitted to a mechanical and enzymatic digestion with collagenase IV. The cell suspension was filtered and centrifuged and the cells obtained were diluted in 15 mL of DMEM-F12 supplemented media. Diluted cells were seeded in 3 culture flasks T25, staying in a controlled environment and changing the medium every 2 days. For the analysis and characterization, the cells were assessed by the Nile red staining to