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Sample records for cell suspension cultures

  1. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    . 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. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

  3. Phosphatidylinositol species of suspension cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Heim, S.; Wagner, K.G.

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with (/sup 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 /sup 3/H-labeled species by autoradiography. The ratio of (/sup 3/H)inositol incorporation into PI, PIP and PIP/sub 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.

  4. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... 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...

  6. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

  7. Auxin requirements of sycamore cells in suspension culture.

    Science.gov (United States)

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

    1983-04-01

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

  8. Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P.tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L-1 2,4-D and 0.05 mg·L-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10 mg·L-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10 mg·L-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

  9. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

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

    Science.gov (United States)

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

    1992-12-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Jan Vitecek; Vilem Reinohl; Russell L.Jones

    2008-01-01

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

  12. 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

  13. Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids

    Science.gov (United States)

    T-87 suspension cell cultures are increasingly used in Arabidopsis research, but there are no reports describing their lipid composition or biosynthesis. To evaluate if T-87 cell cultures as a model system for analysis of lipid metabolism, including tests of gene candidate functions, we have deter...

  14. Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice

    OpenAIRE

    Matsuno, Koya; Fujimura, Tatsuhito

    2014-01-01

    A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA a...

  15. Isolation and culture of protoplasts of Ma-phut (Garcinia dulcis derived from cell suspension culture

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2008-09-01

    Full Text Available Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS medium containing 3% sucrose,1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l benzyladenine (BA and 500 mg/l polyvinylpyrrolidone (PVP, was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4oC. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10 in combination with 1% macerozyme R-10 gave an optimum result in both yield and viability of protoplasts at 5.7x106/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x105/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%. The first division of the protoplasts was obtained after 2 days of culture and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callusformation and plantlet regeneration was not obtained.

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

    Energy Technology Data Exchange (ETDEWEB)

    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{center_dot}L{sup -1} and IAA 0.2 mg{center_dot}L{sup -1} in the dark, and was increased by adding 1 {mu}M Cu{sup 2+} and 100 {mu}M 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{center_dot}L{sup -1} and IAA 0.2 mg{center_dot}L{sup -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 {gamma} radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS ({beta}-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.

  17. 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.

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

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    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 2×10(9) CM/L at scales up to 1L 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.

  19. Putrescine facilitated enhancement of capsaicin production in cell suspension cultures of Capsicum frutescens.

    Science.gov (United States)

    Sudha, Govindaswamy; Ravishankar, Gokare A

    2003-04-01

    Putrescine treatment (0.1 mmol/L) influenced enhancement of growth and capsaicin production in the cell suspension cultures of C. frutescens. The administration of polyamine inhibitor DFMA (alpha-DL-difluoromethylarginine) resulted in a reduction of the growth, capsaicin content and the endogenous titres of polyamines (PAs). The capsaicin synthase activity was also higher in the putrescine (Put) treated cultures. Ethylene levels were lower in the cultures treated with putrescine. This study suggested that Put facilitates growth and capsaicin production.

  20. [Effects of several factors on cell growth and ginsenoside accumulation of Panax ginseng suspension culture].

    Science.gov (United States)

    Li, Tie-Jun; Lian, Mei-Lan; Yu, Dan; Shao, Chun-Hui; Piao, Xuan-Chun

    2013-12-01

    To improve cell suspension culture system of Panax ginseng, the dynamic of cell growth and medium consumption were studied, and the effects of filter on the culture vessel, revolution number, and inoculation density on cell growth and ginsenoside accumulation were also investigated. The maximum cell growth and ginsenoside accumulation was found on the 20th days of suspension culture, therefore, 20 days were confirmed as a suitable culture period for mass production of ginsenoside. Cell growth and ginsenoside content were promoted when the culture vessel had a ventilated filter. Revolution speed during suspension culture affected cell growth, but not ginsenoside content, a peak of ginsenoside productivity was found in the treatment of 120 r x min(-1). Inoculation density also influenced cell growth and ginsenoside accumulation, inoculation density of 6 g was better than other inoculation densities, the ginsenoside content and productivity were up to 12.8 mg x g(-1) DW and 146.6 mg x L(-1), respectively. PMID:24791486

  1. Accumulation of podophyllotoxin and related lignans in cell suspension cultures of Linum album

    NARCIS (Netherlands)

    Smollny, T.; Wichers, H.; Kalenberg, S.; Shahsavari, A.; Petersen, M.; Alfermann, A.W.

    1998-01-01

    Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotox

  2. DIGLUCOSYLATION OF SALICYL ALCOHOL BY CELL SUSPENSION CULTURES OF SOLANUM LACINIATUM

    Institute of Scientific and Technical Information of China (English)

    ACHMAD SYAHRANI; FRANSISCA HARTUTI; GUNAWAN INDRAYANTO; ALISTAIR L.WILKINS

    2001-01-01

    A new biotransformation product, salicyl alcohol-7-O-β-D-(β-l,6-D-glucopyranosyl)-gluco pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

  3. 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.

  4. 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

  5. A simple and efficient method for the long-term preservation of plant cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Boisson Anne-Marie

    2012-01-01

    Full Text Available Abstract Background The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority. Results Sycamore (Acer pseudoplatanus and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed. Conclusion We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.

  6. 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% at the...... start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied 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...

  7. Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

    DEFF Research Database (Denmark)

    Hong, Shin-Young; Kwon, Tae-Ho; Jang, Yong-Suk;

    2006-01-01

    Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using...... particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell...... suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the...

  8. Survival of Suspension-cultured Sycamore Cells Cooled to the Temperature of Liquid Nitrogen.

    Science.gov (United States)

    Sugawara, Y; Sakai, A

    1974-11-01

    Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) which were immersed in liquid nitrogen after prefreezing to the temperatures from -30 to -50 C in the presence of dimethylsulfoxide and glucose as cryoprotective additive could proliferate vigorously when rewarmed rapidly in water at 40 C. For maintaining high viability of the cells after immersion in liquid nitrogen, it seems to be essential to use the cells at the later lag phase or the early cell division phase. This study provides a possibility for long term preservation in liquid nitrogen of plant-cultured lines.

  9. Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

    Science.gov (United States)

    Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

    2016-05-15

    Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. PMID:26658353

  10. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  11. [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.

  12. Purification and Characterization of Abundant Secreted Protein in Suspension-Cultured Pumpkin Cells 1

    Science.gov (United States)

    Esaka, Muneharu; Enoki, Keiko; Kouchi, Bonko; Sasaki, Takuji

    1990-01-01

    The abundant secreted protein with molecular weight of 32,000 was purified from the culture medium of suspension-cultured pumpkin (Cucurbita sp.) cells. Two steps, ammonium sulfate fractionation and Sepharose 6B column chromatography, were sufficient for purification to homogeneity. Antibodies against the pure protein were used to show that a protein of the same size is made by callus cells. There is considerable homology between the amino-terminal amino acid sequence of this secreted protein and chitinase isolated from tobacco (Nicotiana tabacum L.) or bean (Phaseolus vulgaris L.). Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667554

  13. 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.

  14. C-27 AND C-3 GLUCOSYLATION OF DIOSGENIN BY CELL SUSPENSION CULTURES OF COSTUS SPECIOSUS

    Institute of Scientific and Technical Information of China (English)

    GUNAWAN INDRAYANTO; SITI ZUMAROH; ACHMAD SYAHRANI; ALISTAIR L. WILKINS

    2001-01-01

    3-O-[β-D-glucopyranosyl-(l″→ 2′)-β-D-glucopyranosyl], 27-O-β-D-glucopyranosyl-(25R)-spir ost-5-ene-3β,27-diol was isolated from cell suspension cultures of Costus speciosus, following incubation with diosgenin, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C NMR spectral data, and positive and negative ion ESMS spectral data.

  15. 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.

  16. Uptake and metabolism of sugars by suspension-cultured catharanthus roseus cells

    Energy Technology Data Exchange (ETDEWEB)

    Ashihara, Hiroshi; Sagishima, Kyoko; Kubota, Kaoru (Ochanomizu Univ., Tokyo (Japan))

    1989-04-01

    The Uptake and metabolism of sugars by suspension-cultured Catharanthus roseus cells were investigated. Substantially all the sucrose in the culture medium was hydrolyzed to glucose and fructose before being taken up by the cells. The activity of invertase bound to cell walls, determined in situ, was high at the early stage of culture. Glucose was more easily taken up by the cells than was fructose. Tracer experiments using (U-{sup 14}C)glucose and (U-{sup 14}C)fructose indicated that glucose is a better precursor for respiration than fructose, while fructose is preferentially utilized for the synthesis of sucrose, especially in the early phase of cell growth. These results suggest that fructose is utilized for the synthesis of sucrose via the reaction catalyzed by sucrose synthase, prior to the phosphorylation by hexokinase or fructokinase.

  17. Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines.

    Science.gov (United States)

    Shi, Y; Ryu, D D; Ballica, R

    1993-03-25

    Data on viscous (eta') and elastic (eta'') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 microm as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 microm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field.

  18. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  19. 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.

  20. Protein Adsorption Alters Hydrophobic Surfaces Used for Suspension Culture of Pluripotent Stem Cells

    Science.gov (United States)

    Jonas, Steven J.; Stieg, Adam Z.; Richardson, Wade; Guo, Shuling; Powers, David N.; Wohlschlegel, James; Dunn, Bruce

    2015-01-01

    This Letter examines the physical and chemical changes that occur at the interface of methyl-terminated alkanethiol self-assembled monolayers (SAMs) after exposure to cell culture media used to derive embryoid bodies (EBs) from pluripotent stem cells. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy analysis of the SAMs indicates that protein components within the EB cell culture medium preferentially adsorb at the hydrophobic interface. In addition, we examined the adsorption process using surface plasmon resonance and atomic force microscopy. These studies identify the formation of a porous, mat-like adsorbed protein film with an approximate thickness of 2.5 nm. Captive bubble contact angle analysis reveals a shift toward superhydrophilic wetting behavior at the cell culture interface due to adsorption of these proteins. These results show how EBs are able to remain in suspension when derived on hydrophobic materials, which carries implications for the rational design of suspension culture interfaces for lineage specific stem-cell differentiation. PMID:26261952

  1. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed. PMID:21236330

  2. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.

  3. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  4. Histology of embryoid development in oil palm (Elaeis guineensis Jacq. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Songrat Tinnongjig

    2001-11-01

    Full Text Available Embryos of oil palm (Elaeis guineensis Jacq. variety tenera were cultured on Eeuwens or Y3 (1976; 1978 medium supplemented with 2 mg/l 2,4-D. Calluses were initiated from these embryos. The eight-weekold calluses derived from embryos were transferred to modified Y3 liquid medium devoid of 2,4-D and supplemented with NAA, BA and coconut water to establish cell suspension culture. After a period of culture,these cells were then subcultured to the same medium without plant growth regulators to induce embryoid formation. The calluses and embryoids were harvested at various times, fixed, sectioned, stained and examined microscopically. Histological study revealed that embryoid occurred from meristematic cells with dense cytoplasm along the callus clumps.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Ilona Turek

    2015-09-01

    Full Text Available 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. Serum-free spheroid suspension culture maintains high proliferation and differentiation potentials of mesenchymal stem cells

    Science.gov (United States)

    Alimperti, Stella; Wen, Yuan; Lei, Pedro; Tian, Jun; Campbell, Andrew; Andreadis, Stelios T.

    2016-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn’s disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this present study, we developed and optimized serum free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serum-containing adherent cultures. PMID:24616445

  8. 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

  9. 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

  10. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  11. UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselinum hortense

    OpenAIRE

    Kreuzaler, Fritz; Ragg, Hermann; Fautz, Erich; David N Kuhn; Hahlbrock, Klaus

    1983-01-01

    DNAs complementary to poly(A)+ mRNAs from UV-irradiated cell suspension cultures of parsley (Petroselinum hortense) were inserted into pBR322 and used to transform Escherichia coli strain RR1. A clone containing a DNA complementary to chalcone synthase mRNA was identified by hybrid-selected and hybrid-arrested translation. Large and rapid changes in the amount of chalcone synthase mRNA in response to irradiation of the cells was detected by RNA blot hybridization experiments. The pattern of c...

  12. APOPTOSIS AND TAXOL PRODUCTION IN SUSPENSION CULTURES OF Taxus spp.CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    l.lntroductionSuspension cultures of Taxal chinensis var.mairei frequently accumulate Taxol (paclitaxel),which is clinically effective amineoplanic agent.TSXol is known to act by enhancing thePOlymeriZation of tubulin in the initiation andextension of microtubules, ac has been shown toinduce apoptosis in human and animal cellslll.Apoptosis, also known as programmed cell death, isthe active process of cell death which occurs duringdevelopment and in resPOnse tO enviboental cues ofa multicellular organism. In...

  13. Changes of Respiration Activities in Cells of Winter Wheat and Sugar Cane Suspension Cultures During Programmed Cell Death Process

    OpenAIRE

    I.V. Lyubushkina; A.V. Fedyaeva; Stepanov, A.V.; T.P. Pobezhimova

    2015-01-01

    Process of cell death in suspension cultures of winter wheat and sugar cane under high (50 °С) and negative (-8 °С) temperature treatment has been studied. It has been shown, that programmed cell death (PCD) process caused by the negative temperature in the culture of winter wheat was noted for slow rate of realization and it was carried out for 10 days. It has been state that rate of cell respiration was significantly higher than in the control culture. At the same time PCD processes induced...

  14. Fate and metabolism of the brominated flame retardant tetrabromobisphenol A (TBBPA) in rice cell suspension culture.

    Science.gov (United States)

    Wang, Songfeng; Cao, Siqi; Wang, Yongfeng; Jiang, Bingqi; Wang, Lianhong; Sun, Feifei; Ji, Rong

    2016-07-01

    Tetrabromobisphenol A (TBBPA) is the brominated flame retardant with the highest production volume and its bioaccumulation in environment has caused both human health and environmental concerns, however the fate and metabolism of TBBPA in plants is unknown. We studied the fate, metabolites, and transformation of (14)C-labeled TBBPA in rice cell suspension culture. During the incubation for 14 days, TBBPA degradation occurred continuously in the culture, accompanied by formation of one anisolic metabolite [2,6-dibromo-4-(2-(2-hydroxy)-propyl)-anisole] (DBHPA) (50% of the degraded TBBPA) and cellular debris-bound residues (46.4%) as well as mineralization (3.6%). The cells continuously accumulated TBBPA in the cytoplasm, while a small amount of DBHPA (2.1% of the initially applied TBBPA) was detectable inside the cells only at the end of incubation. The majority of the accumulated residues in the cells was attributed to the cellular debris-bound residues, accounting for 70-79% of the accumulation after the first incubation day. About 5.4% of the accumulation was associated with cell organelles, which contributed 7.5% to the cellular debris-bound residues. Based on the fate and metabolism of TBBPA in the rice cell suspension culture, a type II ipso-substitution pathway was proposed to describe the initial step for TBBPA degradation in the culture and balance the fate of TBBPA in the cells. To the best of our knowledge, our study provides for the first time the insights into the fate and metabolism of TBBPA in plants and points out the potential role of type II ipso-hydroxylation substitution in degradation of alkylphenols in plants. Further studies are required to reveal the mechanisms for the bound-residue formation (e.g., binding of residues to specific cell wall components), nature of the binding, and toxicological effects of the bound residues and DBHPA.

  15. Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

    Science.gov (United States)

    Shakhbazau, Antos; Mirfeizi, Leila; Walsh, Tylor; Wobma, Holly M; Kumar, Ranjan; Singh, Bhagat; Kallos, Michael S; Midha, Rajiv

    2016-02-01

    Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

  16. Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays

    International Nuclear Information System (INIS)

    Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study the authors used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts. (Auth.)

  17. Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.

    Science.gov (United States)

    Andreazza, Nathalia Luiza; Abreu, Ilka Nacif; Sawaya, Alexandra Christine Helena Frankland; Mazzafera, Paulo

    2015-03-01

    Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results. PMID:25474486

  18. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  19. Influences of Plant Growth Regulators,Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

    Institute of Scientific and Technical Information of China (English)

    TangWei; WuJiongyuan; 等

    1995-01-01

    A cell suspension culture of Panax ginseng which may be continuously subcultured has been established.Embryogenic callus derived from clutured young leaves was used to initiate the culture,Plant growth regulators,basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development ,The best selection of plant growth regulator,basal medium and carbohydrate level is 2mg/L 2,4-D:0.5mg/L KT,MS and 3% sucrose respectively.

  20. Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    Zhang Dongyan; Yu Fang; Bai Fengwu; An Lijia

    2006-01-01

    The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension cell culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO-3/NH+4, and nitrate was favourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N source. The effect of total initial N on the cell cultures was also investigated with NO-3/NH+4 ratio of 1∶2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

  1. 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.

  2. Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

    Directory of Open Access Journals (Sweden)

    Nikolay Vasilev

    Full Text Available A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

  3. Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

    Science.gov (United States)

    Nagella, Praveen; Dandin, Vijayalaxmi S; Murthy, Hosakatte Niranjana

    2016-01-01

    Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described. PMID:27108321

  4. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    Science.gov (United States)

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  5. 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

    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......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...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...

  6. Establishment of suspension cell culture of Gymnema sylvestre R.Br.- A threatened anti-diabetic plant

    Directory of Open Access Journals (Sweden)

    Karthic Raju

    2012-04-01

    Full Text Available A cell suspension culture was established from leaf explants of wild Gymnema sylvestre plants collected from Muniyankudisai,Tamilnadu, India. Murashige and Skoog medium supplemented with 9.0 μM l-1 of 2, 4- Dichlorophenoxy acetic acid and 2.1 μM l-1Benzyl adenine produced yellow friable callus with green patches.The cells were subcultured conscientiously twelve times to getconsistent growth of the cells in suspension. From the 10thsubculture onwards callus cells acclimatized to grow in suspensionwith aggregation and reached 168.6 g l-1 fw and 5.16 g l-1 dw of cellbiomass.

  7. 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.

  8. Biotransformation of perfumery terpenoids, (−)-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

    OpenAIRE

    Musharraf Syed Ghulam; Naz Sheeba; Najeeb Asma; Khan Saifullah; Choudhary M Iqbal

    2012-01-01

    Abstract Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm) with fungal and plant cell culture. Results Biotransformation of (−)-ambrox (1) with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmal...

  9. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Henning Kempf

    2014-12-01

    Full Text Available To harness the potential of human pluripotent stem cells (hPSCs, an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion. Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity was enabled that were directly applicable to bioartificial cardiac tissue formation.

  10. 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.

  11. 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

    2016-01-01

    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.

  12. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  13. Lipoxygenase activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.).

    Science.gov (United States)

    Kollárová, R; Oblozinský, M; Kováciková, V; Holková, I; Balazová, A; Pekárová, M; Hoffman, P; Bezáková, L

    2014-08-01

    In this study we investigated the influence of biotic elicitor (phytopathogenic fungus Botrytis cinerea) and abiotic elicitors (methyljasmonate [MJ] and salicylic acid [SA]) on lipoxygenase (LOX) activity and sanguinarine production in cell suspension cultures of California poppy (Eschscholtzia californica CHAM.). We have observed different time effects of elicitors (10, 24, 48 and 72 h) on LOX activity and production of sanguinarine in in vitro cultures. All elicitors used in the experiments evidently increased the LOX activity and sanguinarine production in contrast to control samples. The highest LOX activities were determined in samples elicitated by MJ after 48 h and 72 h and the lowest LOX activities (in contrast to control samples) were detected after biotic elicitation by Botrytis cinerea. These activities showed about 50% lower level against the activities after MJ elicitation. The maximal amount of sanguinarine was observed after 48 h in MJ treated cultures (429.91 mg/g DCW) in comparision with control samples. Although all elicitors affect the sanguinarine production, effect of SA and biotic elicitor on sanguinarine accumulation in in vitrocultures was not so significant than after MJ elicitation. PMID:25158577

  14. UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

    Science.gov (United States)

    2010-01-01

    The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase. PMID:20704760

  15. 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.

  16. Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

    2015-04-01

    Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

  17. 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.

  18. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Science.gov (United States)

    Ramani, Shilpa; Chelliah, Jayabaskaran

    2007-01-01

    Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. PMID:17988378

  19. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Chelliah Jayabaskaran

    2007-11-01

    Full Text Available Abstract Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc and strictosidine synthase (Str. In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s, Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

  20. 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.

  1. Xyloglucan biosynthesis by Golgi membranes from suspension-cultured sycamore (Acer pseudoplatanus) cells

    International Nuclear Information System (INIS)

    Xyloglucan is a major hemicellulose polysaccharide in plant cell walls. Biosynthesis of such cell wall polysaccharides is closely linked to the process of plant cell growth and development. Xyloglucan polysaccharides consist of a β-1,4 glucan backbone synthesized by xyloglucan synthase and sidechains of xylose, galactose, and fucose added by other transferase enzymes. Most plant Golgi and plasma membranes also contain glucan synthases I ampersand II, which make β-1,4 and β-1,3 glucans, respectively. All of these enzymes have very similar activities. Cell walls on suspension-cultured cells from Acer pseudoplatanus (sycamore maple) were enzymatically softened prior to cell disruption by passing through a 30 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on top-loaded or flotation sucrose density gradients. Samples were collected by gradient fractionation and assayed for membrane markers and xyloglucan and glucan synthase activities. Standard marker assays (cyt. c reductase for eR, IDPase ampersand UDPase for Golgi, and eosin 5'-malelmide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of 14C-labeled sugars from UDP-glucose and UDP-xylose was used to detect xyloglucan synthase, glucan synthases I ampersand II, and xylosyl transferase in Golgi membrane fractions. These activities overlapped, although distinct peaks of xyloglucan synthase and xylosyl transferase were found. Ca++ had a stimulatory effect on glucan synthases I ampersand II, while Mn++ had an inhibitory effect on glucan synthase I in the presence of Ca++. The similarity of these various synthase activities demonstrates the need for careful structural characterization of newly synthesized polysaccharides

  2. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  3. 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.

  4. Stereo and region-selective biosynthesis of two new dihydroartemisinic acid glycosides by suspension-cultured cells of Artemisia annua

    Directory of Open Access Journals (Sweden)

    Jianhua Zhu

    2014-01-01

    Full Text Available Background: The system of plant-cultured cells is one of the optimal systems to investigate biosynthesis pathway and their bioactive intermediates. Objective: To study the biosynthesis of dihydroartemisinic acid (1 by suspension-cultured cells of Artemisia annua. Materials and Methods: Substrate (compound 1 was administered into the suspension-cultured cells of A. annua and co-cultured for 2 days. The methanol extract was separated on various column chromatography methods and the structures of two biosynthesis products were elucidated based on the analysis of 1 H NMR, 13 C NMR, 2D NMR, and ESI-MS. Time-course curve was also established. Furthermore, in vitro antitumor activities of compounds 1-3 against HepG2, K562, and A549 cell lines were evaluated by MTT assay. Results: Two new compounds were obtained, namely 3α-hydroxy-dihydroartemisinic acid-α-D - glucopyranosyl ester (2 and 15-hydroxy-cadin-4-en-12-oic acid-β-d - glucopyranosyl ester (3. The results demonstrated that the cultured cells of A. annua possessed the abilities to stereo-selective hydroxylate and region-selective glycosylate sesquiterpene compounds in a highly efficient manner. Inhibitory effects of compounds 1-3 on proliferation of HepG2, K562, and A549 cell lines in vitro were also investigated. Conclusion: Two new dihydroartemisinic acid glycosides were obtained by stereo- and region-selective biosynthesis with cultured cells of A. annua.

  5. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells. PMID:24141419

  6. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  7. Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

    Institute of Scientific and Technical Information of China (English)

    Dai Huanqin; Lu Cunfu; Zhang Hui; Zhang Xujia

    2003-01-01

    Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of-17.5 ℃) in cell suspension at 4-5 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of-12.5 ℃ in nonacclimated cells to LT50 of-17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.

  8. Physical modeling of animal cell damage by hydrodynamic forces in suspension cultures.

    Science.gov (United States)

    Lu, G Z; Gray, M R; Thompson, B G

    1992-12-01

    Physical damage of animal cells in suspension culture, due to stirring and sparging, is coupled with complex metabolic responses. Nylon microcapsules, therefore, were used as a physical model to study the mechanisms of damage in a stirred bioreactor and in a bubble column. Microcapsule breaskage folowed first-order kinetices in all experiments Entrainment of bubbles into the liquid phase in the stirred bioreactor gave more microcapsule breakage. In the bubble column, the bubble bursting zone at gas-liquid interface was primarily responsible for microcapsule breakage. The forces on the microcapsules were equivalent to an external pressure of approximately 4 x 10(4) N. m(-2), based on the critical microcapsule diameter for survival of 190 microm. A stable foam layer, however, was found to be effective in protecting microcapsules from damage. The microcapsule transport to the gas-liquid interface and entrainment into the foam phase was consistent with flotation by air bubbles. This result implies that additives and operation of bioreactors should be selected to minimize flotation of cells. PMID:18601080

  9. Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells.

    Science.gov (United States)

    Stevenson, T T; McNeil, M; Darvill, A G; Albersheim, P

    1986-04-01

    The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.

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

    Science.gov (United States)

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

    2009-12-01

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

  11. Establishment of Suspension Cell Culture from Agrobacterium-transformed Hairy Root Cells of Psammosilene tunicoides, an Endangered and Rare Medicinal Plant of China

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

    Full Text Available Psammosilene tunicoides is an important medicinal plant endemic in China. Its annual yield is severely limited due to slow growth, poor seed germination and excessive collection. To satisfy the growing market demands, it’s necessary to seek alternatives to field cultivation and wild resources of this endangered plant. Using Agrobacterium -transformed hairy roots as initial explants, here, we reported the development of a suspension cell culture system for P. tunicoides. Results showed the Agrobacterium -transformed hairy roots-derived suspension cells are fast in growth and strong in capacity for accumulation of bioactive metabolites. We established that 1/2MS was a suitable medium for culturing the hairy root-derived suspension cells and the optimal combination of phytohormones is 1.5 mg/L 2, 4-D+0.5 mg/L 6-BA+0.25 mg/L NAA+0.1 mg/L KT. Under this condition, the maximal biomass was achieved at the 20th day of culture with an average growth rate of 0.72 g/L/d; and the intracellular saponine content reached 0.92%, comparable to that of mother hairy roots. Compared with the normal P. tunicoides suspension cells, the hairy roots-derived suspension cells exhibited features of fast growth, short culture period and high concentration of saponines, suggesting that the large scale culture of hairy root-derived cells could be a feasible alternative to the wild resources of P. tunicoides.

  12. In vitro induction of α-pinene, pulegone, menthol, menthone and limonene in cell suspension culture of pennyroyal (Mentha pulegium).

    Science.gov (United States)

    Darvishi, E; Kahrizi, D; Bahraminejad, S; Mansouri, M

    2016-01-01

    Medicinal plants are known as important sources of secondary metabolites. Because of the economic value of pennyroyal [Mentha pulegium L. (Lamiaceae)] in food industries, propagation of this valuable plant has special importance. Plant cell suspension culture can increase some produced components. The aim of this research was performing cell culture for induction of some secondary metabolites of M. pulegium and compares it with native one. The MS medium was used for suspension culture. To investigate quantitative materials, 4 levels of yeast extract elicitor (20, 40, 60 and 80 mg/L) and salicylic acid in 4 levels (2, 4, 6 and 8 mg/L) were used. Obtained extracts were analyzed by GC-MS. Statistical analysis showed that the amount of limonene, menthone, menthol and α-pinene were more than mentioned compounds in natural plant as control. The maximum amount of this metabolites were obtained as limonene (in 60 mg/l yeast extract), menthone (in 40 mg/l yeast extract and 2 mg/l salicylic acid), menthol (in 6 mg/l salicylic acid) and α-pinene (in 4 mg/l salicylic acid) in the M. pulegium cell culture. The Pulegone was fond more in natural plants than cell culture mass. The most important secondary metabolites were increased by cell culture containing of salicylic acid and yeast extract elicitors in M. pulegume. PMID:27064866

  13. 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.

  14. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    PLANT CELL WALLS SERVE SEVERAL FUNCTIONS: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.

  15. Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

    Institute of Scientific and Technical Information of China (English)

    李景川; 马忠海; 元英进; 孙安慈; 胡昌序

    2001-01-01

    The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of “partition” and “bifurcation” were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

  16. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    Science.gov (United States)

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  17. Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    Yang Shengchang

    2006-01-01

    A rapid and concentration-dependent generation of superoxide anion (·O-2), measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts (LaCl3 and GdCl3) were added to tobacco (Nicotiana tabacum) cell suspension culture.Addition of superoxide dismutase (480 U·ml-1) and Tiron (5 μmol·L-1) to cell culture suspension decreases the level of lanthanide cation-induced ·O-2 generation, suggesting that ·O-2 generation is extra-cellular.Pretreatment of the cell culture suspension with diphenyleneiodonium (10 and 50 μmol·L-1), quinacrine (1 and 5 mmol·L-1) and imidazol (10 mmol·L-1), inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase.In addition, addition of SHAM (1 and 5 mmol·L-1), azide (0.2 and 1 mmol·L-1), inhibitor of peroxidase, has no influence on ·O-2 generation.

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

    Directory of Open Access Journals (Sweden)

    Francesca Rizzello

    2014-10-01

    Full Text Available 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.

  19. A method for rapid preparation of single-cell suspensions from rat hepatocyte primary cultures on collagen substratum and the mechanism of cell dissociation.

    Directory of Open Access Journals (Sweden)

    Miyazaki,Masahiro

    1988-12-01

    Full Text Available A method has been developed for the rapid preparation of single-cell suspensions from rat hepatocyte primary cultures on collagen substratum. Hepatocytes were adequately dissociated into single cells when the cultures were first treated with a combination of trypsin and ethylenediaminetetraacetic acid (EDTA and then with collagenase. However, when the order was reversed, hepatocytes were inadequately dispersed. The possible mechanism of cell dissociation is discussed on the basis of the experimental data obtained.

  20. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Kajani Abolghasem

    2012-10-01

    Full Text Available Abstract Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale. Methods We investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing. Results The yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  1. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Abolghasem Abbasi Kajani

    2012-10-01

    Full Text Available Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale.MethodsWe investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing.ResultsThe yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  2. Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

    Science.gov (United States)

    Haas, Christiane; Hegner, Richard; Helbig, Karsten; Bartels, Kristin; Bley, Thomas; Weber, Jost

    2016-06-01

    Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc. PMID:26614913

  3. A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnanajothi; Jeyaraj, Murugaraj; Rajesh, Manoharan; Muthuselvam, Manickam; Selvaraj, Natesan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-08-01

    Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.

  4. 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

    . The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival...... 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...

  5. 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.

  6. 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

    2016-08-01

    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 (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 by tumor

  7. In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

    Indian Academy of Sciences (India)

    S Sujanya; B Poornasri Devi; Isha Sai

    2008-03-01

    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, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate:ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

  8. 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. PMID:25064634

  9. Cell Suspension Culture-Mediated Incorporation of the Rice Bel Gene into Transgenic Cotton

    OpenAIRE

    Liping Ke; RuiE Liu; Bijue Chu; Xiushuang Yu; Jie Sun; Brian Jones; Gang Pan; Xiaofei Cheng; Huizhong Wang; Shuijin Zhu; Yuqiang Sun

    2012-01-01

    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 liqu...

  10. Establishing in vitro Zinnia elegans cell suspension culture with high tracheary elements differentiation

    NARCIS (Netherlands)

    Twumasi, P.; Schel, J.H.N.; Ieperen, van W.; Woltering, E.J.; Emons, A.M.C.

    2009-01-01

    The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and f

  11. Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis.

    OpenAIRE

    Borderies, Gisèle; Jamet, Elisabeth; Lafitte, Claude; Rossignol, Michel; Jauneau, Alain; Boudart, Georges; Monsarrat, Bernard; Esquerré-Tugayé, Marie-Thérèse; Boudet, Alain; Pont-Lezica, Rafael

    2003-01-01

    The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked...

  12. 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)

    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)

  13. 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. PMID:21556691

  14. Interaction between abscisic acid and nitric oxide in PB90-induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures.

    Science.gov (United States)

    Chen, Qian; Chen, Zunwei; Lu, Li; Jin, Haihong; Sun, Lina; Yu, Qin; Xu, Hongke; Yang, Fengxia; Fu, Mengna; Li, Shengchao; Wang, Huizhong; Xu, Maojun

    2013-01-01

    Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells. PMID:23554409

  15. Large-scale production of monoclonal antibodies in suspension culture.

    Science.gov (United States)

    Backer, M P; Metzger, L S; Slaber, P L; Nevitt, K L; Boder, G B

    1988-10-01

    Monoclonal antibodies are being manufactured for clinical trials in suspension culture at the 1300-L scale. Suspension culture offers some advantages relative to high-density mammalian cell culture methods; in particular, the ability to closely monitor the behavior of cells in a homogeneous environment. Computer control and on-line mass spectrography of exit gases provide instantaneous information about the culture metabolic activity. Air sparging and agitation by marine impeller provide aeration sufficient to maintain a constant dissolved oxygen tension at cell concentrations up to 5.0 x 10(6) cells/mL without causing apparent cell damage.

  16. Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Baroja-Fernández, E; Zandueta-Criado, A; Rodríguez-López, M; Akazawa, T; Pozueta-Romero, J

    2000-09-01

    The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.

  17. Im"plant"ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation.

    Science.gov (United States)

    Kajiura, Hiroyuki; Fujiyama, Kazuhito

    2015-01-01

    Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

  18. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  19. 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.

  20. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

    Directory of Open Access Journals (Sweden)

    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  1. Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins.

    Science.gov (United States)

    Yamazaki, N; Fry, S C; Darvill, A G; Albersheim, P

    1983-07-01

    A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

  2. INSECTICIDAL POTENTIALITY OF FLAVONOIDS FROM CELL SUSPENSION CULTURE OF MARCHANTIA LINEARIS LEHM. & LINDENB AGAINST SPODOPTERA LITURA F.

    Directory of Open Access Journals (Sweden)

    Remya Krishnan

    2015-05-01

    Full Text Available Bryophytes were diverse, primitive non vascular am phibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing gr owth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lowe r rpm bring about lower leve l of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quer cetin, luteolin, apigenin , rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedan t, larvicidal and pupicidal activities at all the concentrations against 5 th instar larvae of Spodoptera litura . The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food and ECD (Efficiency of conversion of digested food and increased AD (Approximate digestibility and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the co nsumption of the control diet by the larvae. Faecal production reduced proportionally with

  3. [Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

    Science.gov (United States)

    Wei, Yue-Rong; Huang, Xue-Lin; Li, Jia; Huang, Xia; Li, Zhe; Li, Xiao-Ju

    2005-01-01

    Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV

  4. A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    张卫; Shintaro; Furusaki; Chris; Franco

    1999-01-01

    A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30℃ (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthoeyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was deereased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influeneed by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg(?)g-fresh cell-1 was obtained on day 9 by a temperature-

  5. Identification and quantitative determination of pinoresinol in Taxus ×media Rehder needles, cell suspension and shoot cultures

    Directory of Open Access Journals (Sweden)

    Paulina Mistrzak

    2015-01-01

    Full Text Available The aim of our study was to investigate the presence and quantitative contents of lignans in the tissues of Taxus ×media. The presence of the lignans: pinoresinol, matairesinol and secoisolariciresinol was assessed in needles, shoots cultures and suspension culture. Pinoresinol was the only lignan found in the tissue of T. ×media. The total pinoresinol content in the needles and in the shoots was 1.24 mg/g dry weight (dw and 0.69 mg/g dw, respectively. Most of the pinoresinol identified was appeared glycosidically bound. In needles, the amount of glycosidically bound pinoresinol (0.81 mg/g dw was about twice as high as that of free pinoresinol (0.43 mg/g dw. The content of free and glycosidically bound pinoresinol showed the level of 0.18 mg/g dw and 0.51 mg/g dw, respectively in the in vitro shoot cultures. In the cell culture, no pinoresinol was found.

  6. 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.

  7. Enhancement of vindoline and vinblastine production in suspension-cultured cells of Catharanthus roseus by artemisinic acid elicitation.

    Science.gov (United States)

    Liu, Jinwei; Zhu, Jianhua; Tang, Le; Wen, Wei; Lv, Shuangshuang; Yu, Rongmin

    2014-01-01

    Elicitation is an important strategy to improve production of secondary metabolites in vitro. Artemisinic acid was studied as a novel elicitor to enhance the yield of terpenoid indole alkaloids in the present paper. Our results demonstrated that the concentrations of vindoline and vinblastine were increased by sixfold and twofold, respectively, compared to those of the control group after treatment with artemisinic acid. To elucidate the underlying mechanism, we investigated the gene expression of four enzymes involved in the biosynthetic pathway of vinblastine in the suspension-cultured cells of Catharanthu sroseus. RT-PCR experiment showed that artemisinic acid was able to up-regulate the transcriptions of tryptophan decarboxylase, geraniol 10-hydroxylase, tabersonine 16-hydroxylase and deacetoxyvindoline 4-hydroxylase. PMID:23864440

  8. Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Macherel, D; Kobayashi, H; Akazawa, T

    1988-01-01

    Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

  9. 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

  10. 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.

  11. Establishment and characterization of a Satureja khuzistanica Jamzad (Lamiaceae) cell suspension culture: a new in vitro source of rosmarinic acid.

    Science.gov (United States)

    Sahraroo, Amir; Mirjalili, Mohammad Hossein; Corchete, Purificación; Babalar, Mesbah; Fattahi Moghadam, Mohammad Reza

    2016-08-01

    An in vitro approach to the production of rosmarinic acid (RA), a medicinally important caffeic acid ester, in a cell suspension culture (CSC) of Satureja khuzistanica Jamzad (Lamiaceae) has been investigated for the first time. The CSC was established from friable calli derived from shoot tip explants in Gamborg's B5 liquid medium supplemented with 30 g/L sucrose, 20 mg/L L-glutamine, 200 mg/L casein hydrolysate, 5 mg/L benzyladenine (BA) and 1 mg/L indole-3-butyric acid (IBA). The effect of nitrogen source (KNO3 and (NH4)2SO4) and their different concentrations on the fresh and dry weight (g/L), as well as RA content (mg/g dry weight) were measured. CSC growth measurements indicated a maximum specific cell growth rate of 1.5/day, a doubling time of 7.6 days and a high percentage of cell viability (96.4 %) throughout the growth cycle. Maximum cell fresh weight (353.5 g/L), dry weight (19.7 g/L) and RA production (180.0 mg/g) were attained at day 21 of culture. Cell growth and RA content were affected by nitrogen deficiency. Media containing 8.3 mM of total nitrogen (¼ of B5 standard medium) led to a minimum cell fresh weight (243.0 g/L), dry weight (17.4 g/L) and RA content (38.0 mg/g) after 21 days. The established CSC provided useful material for further optimization experiments aimed at a large-scale production of RA. PMID:26264595

  12. Studies on suspension culture of virginia mallow

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    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.

  13. 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. PMID:27457992

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

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    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.

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

    Science.gov (United States)

    Ke, Liping; Liu, RuiE; Chu, Bijue; Yu, Xiushuang; Sun, Jie; Jones, Brian; Pan, Gang; Cheng, Xiaofei; Wang, Huizhong; Zhu, Shuijin; Sun, Yuqiang

    2012-01-01

    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. PMID:22768325

  16. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  17. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-01

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth. PMID:24773089

  18. Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

    Science.gov (United States)

    Hamerski, D; Schmitt, D; Matern, U

    1990-01-01

    Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

  19. 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

  20. Site of Clomazone Action in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [14C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [14C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID:16667768

  1. Biotransformation of perfumery terpenoids, (−-ambrox® by a fungal culture Macrophomina phaseolina and a plant cell suspension culture of Peganum harmala

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    Musharraf Syed Ghulam

    2012-08-01

    Full Text Available Abstract Background Biotransformation offers chemo enzymatic system to modify the compounds into their novel analogues which are difficult to synthesize by chemical methods. This paper describes the biotransformational studies of ambrox, one of the most important components of natural Ambergris (wale sperm with fungal and plant cell culture. Results Biotransformation of (−-ambrox (1 with a fungal cell culture of Macrophomina phaseolina and a plant cell suspension cultures of Peganum harmala yielded oxygenated products, 3β-hydroxyambrox (2, 6β-hydroxyambrox (3, 1α-hydroxy-3oxoambrox (4, 1α,3β-dihydroxyambrox (5, 13,14,15,16-tetranorlabdane-3-oxo-8,12-diol (6, 3-oxoambrox (7, 2α-hydroxyambrox (8, 3β-hydroxysclareolide (9, and 2α,3β-dihydroxyambrox (10. Metabolite 4 was found to be new compound. These metabolites were structurally characterized on the basis of spectroscopic studies. Conclusion Nine oxygenated metabolites of (−-ambrox (1 were obtained from Macrophomina phaseolina and Peganum harmala. Enzymatic system of screened organisms introduced hydroxyl and keto functionalities at various positions of compound 1 in a stereo- and regio-controlled manner.

  2. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-alpha-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO(3)-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na(2)CO(3) at 1 degrees C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.

  3. Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Mitsui, T; Akazawa, T

    1986-12-01

    Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

  4. Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells).

    Science.gov (United States)

    Wissing, J. B.; Kornak, B.; Funke, A.; Riedel, B.

    1994-01-01

    Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %. PMID:12232252

  5. Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production.

    Science.gov (United States)

    Chu, Chia; Lugovtsev, Vladimir; Golding, Hana; Betenbaugh, Michael; Shiloach, Joseph

    2009-09-01

    MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs. PMID:19706449

  6. Fractal morphology of Beta vulgaris L. cell suspension culture permeabilized with Triton X-100®

    Science.gov (United States)

    Arenas-Ocampo, M.; Alamilla-Beltrán, L.; Vanegas-Espinoza, P. E.; Camacho-Díaz, B. H.; Campos-Mendiola, R.; Gutiérrez-López, G.; Jiménez-Aparicio, A.

    2012-02-01

    In this work, morphology of Beta vulgaris L. cells permeabilized with 0.7mM of Triton X-100® was evaluated using digital image processing and concepts of fractal dimension (perimeter- area relations). Important morphometric changes were found when the contact-time with chemical agent was increased. The size of cells decreased, the cells lost the roundness and their shape was more sinuous; this behaviour was a result of a probable shrinkage caused by the excess of exposure with the permeabilization agent. Morphology of B. vulgaris cells after permeabilization, exhibited a fractal nature since the slope of the ratio of the logarithm of the perimeter vs logarithm of the area was higher than unit. Fractal geometry of the cell morphology was affected as a result of the exposure to Triton X-100®. Those changes can be attributed to the loss of turgor and structure of the cell wall.

  7. Molecular Role of Nitric Oxide in Secondary Products Production in Ginkgo biloba Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Hossam Saad EL-BELTAGI

    2015-04-01

    Full Text Available Effects of sodium nitroprusside (SNP; nitric oxide donor treatment on the enhancement of secondary metabolites production, oxidative stress mediators (O2-. accumulation and antioxidant defense enzymes of Ginkgo biloba callus culture was investigated. On one hand, the obtained data showed a highly metabolic modification of chemical constituents, PAL activity and various antioxidant defense enzymes (APX, SOD, which gradually increased in response to SNP treatments. On the other hands the high NO levels significantly increased the accumulation of various oxidative burst of O2-.. MS basal medium supplemented with casein hydrolase (500 mg/L, NAA and BA at equal concentration (0.5 mg/L recorded the highest number of regenerated shoots (4.81 cm and shoot height (4.96 cm as well as root number (2.25 cm and root length (4.5 cm. The highest survival (40 % was shown in acclimatization on the mixture containing sand, peat moss and vermiculite (1: 1: 1, v/v/v, which significantly confirmed and reflected the variation in survival percentage. Meanwhile, higher treatment (500 μM of NO positively enhanced secondary products accumulation of total tannins, saponins, phenols and total flavonoids in G. biloba callus culture.

  8. SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

    Science.gov (United States)

    This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

  9. Biochemical and morphological responses to abiotc elicitor chitin in suspension-cultured sugarcane cells

    Directory of Open Access Journals (Sweden)

    Maria Izabel Gallão

    2010-04-01

    Full Text Available Cells of Saccharum officinarum submitted to hydrolyzated chitin for 1 to 8h produced phenolic compounds. These alterations were observed through cytochemical methods using Toluidine Blue and Phloroglucinol/HCl. After 4 h, besides cell wall change, there was a change in nuclear pattern of chitin treated cells. There was a 96% increase in nuclear area in 6 h chitin treated material, as observed by Feulgen reaction. The treated cells showed chromatin compacted regions and a degeneration process of nucleoli. In the outer areas of cell wall, there was a polysaccharide desagregation, confirming results obtained for different plants with the use of other elicitors. Peroxidase activity was maximal after 4 h and decreased progressively. PAL activity started to increase at 4 h of incubation. These results showed that chitin hydrolyzate stimulated a defense response in sugarcane cells.Células de Saccharum officinarum quando submetidas a quitina hidrolisada por 1 a 8h produziram material fenólico. Essas alterações foram observadas por meio de métodos citoquímicos como o Azul de Toluidina e Floroglucinol/HCl. Após 4 h, além das mudanças nas paredes celulares houve uma mudança no padrão nuclear das células tratadas com quitina. Por observação da reação de Feulgen, houve um aumento de 96% na área nuclear no material em 6h. Para as células tratadas foram observadas regiões de cromatina compactada e um processo de degeneração do nucléolo. Nas áreas externas da parede celular existia uma desagregação dos polisacarídios confirmando os resultados obtidos para diferentes plantas com o uso de outros elicitores. A atividade da peroxidase foi maxima após 4 h e então decresceu progressivamente. A atividade da PAL aumentou a partir de 4 h de incubação. Estes resultados mostram que o hidrolisado de quitina estimula as respostas de defesa em células de cana.

  10. Regioselective biotransformation of valencene in cell suspension cultures of Citrus sp.

    Science.gov (United States)

    Drawert, F; Berger, R G; Godelmann, R

    1984-02-01

    Three out of five cultivars of citrus species tested convert exogenous valencene via the 2-hydroxy-derivative (nootkatol) to nootkatone. The effect of various valencene concentrations and the time course of the biotransformation were examined. The transformation capability of the cells runs parallel with growth up to the middle of the logarithmic phase and remains constant until the carbon source is completely exhausted. PMID:24253336

  11. 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.

  12. Jasmonic acid effect on the fatty acid and terpenoid indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus.

    Science.gov (United States)

    Goldhaber-Pasillas, Guitele Dalia; Mustafa, Natali Rianika; Verpoorte, Robert

    2014-01-01

    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. PMID:25029072

  13. Factors Influencing beta-Glucan Synthesis by Particulate Enzymes from Suspension-Cultured Lolium multiflorum Endosperm Cells.

    Science.gov (United States)

    Henry, R J; Stone, B A

    1982-03-01

    Particulate enzymes from suspension-cultured ryegrass (Lolium multiflorum Lam.) endosperm cells incorporated glucosyl residues from UDP-glucose and GDP-glucose into beta-glucans. Three types of beta-glucans were produced from UDP-glucose: 1,3-beta-glucan; 1,4-beta-glucan; and mixed-linkage 1,3;1,4-beta-glucan. As in other systems, relatively more 1,4-beta-glucan was produced from a low (10 micromolar) UDP-glucose concentration, and relatively more 1,3-beta-glucan was produced from a high (1 millimolar) UDP-glucose concentration. However, in ryegrass, 1,3;1,4-beta-glucan represented a major proportion of the products at both low and high UDP-glucose concentrations. The arrangement of linkages in the 1,3;1,4-beta-glucan was different at the two concentrations; at the low UDP-glucose concentration, more sequences of three consecutive 1,4-linkages were produced.The effects of pH, temperature, and metal ion concentrations on incorporation were dependent on the UDP-glucose concentration. At the low UDP-glucose concentration, incorporation into all three types of beta-glucan increased with increasing pH. At the high UDP-glucose concentration, 1,3-beta-glucan was the major product at pH 7 and below; 1,4-beta-glucan synthesis was optimal at pH 8; and synthesis of 1,3;1,4-beta-glucan was greatest above pH 8.With 10 micromolar GDP-glucose as substrate, 1,4-beta-glucan, but no 1,3;1,4-beta-glucan, was produced. Incorporation from either UDP-glucose or GDP-glucose was not influenced by the presence of the other. PMID:16662263

  14. Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

    Science.gov (United States)

    Sakano, Katsuhiro; Yazaki, Yoshiaki; Mimura, Tetsuro

    1992-01-01

    Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2′,7′-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested. ImagesFigure 1 PMID:16668939

  15. Metabolic changes of salicylic acid-elicited Catharanthus roseus cell suspension cultures monitored by NMR-based metabolomics

    OpenAIRE

    Mustafa, Natali Rianika; Kim, Hye Kyong; Choi, Young Hae; Verpoorte, Robert

    2009-01-01

    The effect of salicylic acid (SA) on the metabolic profile of Catharanthus roseus suspension cells throughout a time course (0, 6, 12, 24, 48 and 72 h after treatment) was investigated using NMR spectroscopy and multivariate data analysis. When compared to control cell lines, SA-treated cells showed a high level of sugars (glucose and sucrose) up to 48 h after treatment, followed by a dynamic change in amino acids, phenylpropanoids, and tryptamine. Additionally, one compound—2,5-dihydroxybenz...

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

    Science.gov (United States)

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

    2015-09-01

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

  17. 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

  18. Hydrogen sulfide donor sodium hydrosulfide-induced heat tolerance in tobacco (Nicotiana tabacum L) suspension cultured cells and involvement of Ca(2+) and calmodulin.

    Science.gov (United States)

    Li, Zhong-Guang; Gong, Ming; Xie, Hong; Yang, Lan; Li, Jing

    2012-04-01

    Hydrogen sulfide (H(2)S) is considered as a new emerging cell signal in higher plants. Hydrogen sulfide donor, sodium hydrosulfide, pretreatment significantly increased survival percentage of tobacco suspension cultured cells under heat stress and regrowth ability after heat stress, and alleviated decrease in vitality of cells, increase in electrolyte leakage and accumulation of malondialdehyde (MDA). In addition, sodium hydrosulfide-induced heat tolerance was markedly strengthened by application of exogenous Ca(2+) and its ionophore A23187, respectively, while this heat tolerance was weakened by addition of Ca(2+) chelator ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), plasma membrane channel blocker La(3+), as well as calmodulin (CaM) antagonists chlorpromazine (CPZ) and trifluoperazine (TFP), respectively, but intracellular channel blocker ruthenium red (RR) did not. These results suggested that sodium hydrosulfide pretreatment could improve heat tolerance in tobacco suspension cultured cells and the acquisition of this heat tolerance requires the entry of extracellular Ca(2+) into cells across the plasma membrane and the mediation of intracellular CaM.

  19. 气体成分对植物细胞悬浮培养的影响%Effects of gaseous components in plant cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    周煜; 刘涤; 胡之璧

    2001-01-01

    气体成分对植物悬浮培养细胞的生长和次生代谢物的产量有深刻的影响。就有关氧、二氧化碳、乙烯和一些未知成分作用的研究进行了综述。%Gaseous components, including oxygen, carbon dioxide, ethylene and some unidentified gaseous metabolites are proved to have profound effects on the growth and secondary metabolites' accumulation of plant cell suspension cultures. Wider investigation is needed in this area to optimize cultivation strategies for improving production.

  20. Putting the Spotlight Back on Plant Suspension Cultures

    Science.gov (United States)

    Santos, Rita B.; Abranches, Rita; Fischer, Rainer; Sack, Markus; Holland, Tanja

    2016-01-01

    Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)—the first licensed recombinant pharmaceutical protein derived from plants—is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon. PMID:27014320

  1. Putting the spotlight back on plant suspension cultures

    Directory of Open Access Journals (Sweden)

    Rita B. Santos

    2016-03-01

    Full Text Available Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso® – the first licensed recombinant pharmaceutical protein derived from plants – is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plants cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.

  2. Cell suspension method to improve green spot in in-vitro culture of jarak pagar (Jatropha curcas L ) mutant lines

    International Nuclear Information System (INIS)

    Jatropha curcas has a high potential as an alternative energy source, since it can produce natural oil which could be processed into fuel replacing fossil energy. Increasing demand of biodiesel has resulted in increasing demand for high quality of Jatropha germplasm. Cell suspension method is expected to assure the production of a homogeneous germplasm of Jatropha. A laboratory experiment was conducted to evaluate the effectiveness cell suspension method in of Jatropha curcas cotyledon. The explant used in this experiment was Jatropha curcas seed mutant line (JH-38) which has superiority in plant height, early maturity and unseasonable fruiting. Two kinds of in-vitro medium were used for callus induction, i.e. medium A (MS + 2,4-D 2.0 mg/l + BAP 0.5 mg/l + malt extract 0.1 g + agar 8.0 g/l) and medium B (MS + 2,4-D 3.0 mg/l + BAP 0,5 mg/l + malt extract 0,1 g + agar 8.0 g/l). The same medium composition without agar was used for cell generating, and medium ECS (MS + glutamine 0.5 g + casein hydrolysate 0.5 g + IAA 0.5 mg/l + BAP 3.0 mg/l + agar 8.0 g/l for cell growth. Results of the experiment showed that the optimum growth of calli was obtained by explant JH-38/3 in medium A. The growth level of embryonic cell ranged from 0 to 130 %. The optimum percentage green spot is shown by JH-38/1 explant in medium A. (author)

  3. Cell Docking, Movement and Cell-Cell Interactions of Heterogeneous Cell Suspensions in a Cell Manipulation Microdevice

    OpenAIRE

    Long-Sun Huang; Yu-Hung Wang; Yu-Wei Chung; Fei-Lung Lai; Shiaw-Min Hwang

    2011-01-01

    This study demonstrates a novel cell manipulation microdevice for cell docking, culturing, cell-cell contact and interaction by microfluidic manipulation of heterogeneous cell suspensions. Heterogeneous cell suspensions include disparate blood cells of natural killer cells and leukemia cancer cells for immune cell transplantation therapy. However, NK cell alloreactivity from different healthy donors present various recovery response levels. Little is still known about the interactions and cyt...

  4. Functional compartmentation of the Golgi apparatus of plant cells : immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells.

    Science.gov (United States)

    Zhang, G F; Staehelin, L A

    1992-07-01

    The Golgi apparatus of plant cells is engaged in both the processing of glycoproteins and the synthesis of complex polysaccharides. To investigate the compartmentalization of these functions within individual Golgi stacks, we have analyzed the ultrastructure and the immunolabeling patterns of high-pressure frozen and freeze-substituted suspension-cultured sycamore maple (Acer pseudoplatanus L.) cells. As a result of the improved structural preservation, three morphological types of Golgi cisternae, designated cis, medial, and trans, as well as the trans Golgi network, could be identified. The number of cis cisternae per Golgi stack was found to be fairly constant at approximately 1, whereas the number of medial and trans cisternae per stack was variable and accounted for the varying number of cisternae (3-10) among the many Golgi stacks examined. By using a battery of seven antibodies whose specific sugar epitopes on secreted polysaccharides and glycoproteins are known, we have been able to determine in which types of cisternae specific sugars are added to N-linked glycans, and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The beta-1,4-linked d-glucosyl backbone of xyloglucan is synthesized in trans cisternae, and the terminal fucosyl residues on the trisaccharide side chains of xyloglucan are partly added in the trans cisternae, and partly in the trans Golgi network. In contrast, the polygalacturonic/rhamnogalacturonan-I backbone is assembled in cis and medial cisternae, methylesterification of the carboxyl groups of the galacturonic acid residues in the polygalacturonic acid domains occurs mostly in medial cisternae, and arabinose-containing side chains of the polygalacturonic acid domains are added to the nascent polygalacturonic acid/rhamnogalacturonan-I molecules in the trans cisternae. Double labeling experiments demonstrate that xyloglucan and polygalacturonic acid

  5. 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.

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

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    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(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 [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone 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 (Abutilon theophrasti Medic.) 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.

  7. Uptake and Metabolism of Clomazone in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    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 I50 values for growth, chlorophyll (Chl), β-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 [14C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone 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 (Abutilon theophrasti Medic.) 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. PMID:16667349

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

    Energy Technology Data Exchange (ETDEWEB)

    Kwiatkowska, Aleksandra, E-mail: A.Kwiatkows@gmail.com [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Zebrowski, Jacek [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Oklejewicz, Bernadetta [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland); Czarnik, Justyna [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Halibart-Puzio, Joanna [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Wnuk, Maciej [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland)

    2014-05-02

    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.

  9. Bioactive metabolite production and stress-related hormones in Devil's claw cell suspension cultures grown in bioreactors.

    Science.gov (United States)

    Georgiev, Milen; Ludwig-Müller, Jutta; Weber, Jost; Stancheva, Nina; Bley, Thomas

    2011-03-01

    In a previous report, we showed that cell cultures of Harpagophytum procumbens, a South African plant with high medicinal value, accumulate high amounts of anti-inflammatory phenylethanoid glycosides during cultivation in shake-flasks. The aim of the present study was to transfer the phenylethanoid biosynthetic process to a 3-L stirred tank reactor and a 1-L glass-column bioreactor (operated with pulsed aeration). We found that, with stepwise increases in aeration, the stirred tank reactor yielded similar productivities of verbascoside (the major phenylethanoid glycoside in the cells) to those reported for shake-flask cultures (55.68 vs. 54.78 mg verbascoside/L/day, respectively). Transfer in the pulse-aerated column reactor resulted in 165.42 mg verbascoside/L/day, one of the highest yields reported to date. Further, to evaluate the physiological status of the suspended cells in the bioreactors cultures, we examined their hormone levels and compared them to those of cells in shake-flask cultures. While indole-3-acetic acid levels did not differ significantly between the bioreactor and shake-flask cultures, there were considerable differences in their levels of abscisic, jasmonic, and salicylic acids. These results are discussed with respect to relative stress levels in the different cultivation systems. PMID:21104241

  10. Characterization of carrot cell lines resistant to 5-methyltryptophan obtained by irradiating suspension cultures with UV-light

    International Nuclear Information System (INIS)

    A mutagenic procedure of carrot cell suspension by means of UV-light has been established. The application of this procedure to the selection of cell lines resistant to 5-methyltryptophan (5MT) increased 11 times the spontaneous mutation rate. Eighteen colonies selected in the course of one experiment have been analyzed for quantitative resistance to the analogue. Four of the most 5MT-resistant lines selected (one spontaneous and three induced) were also tested for their resistance to azetidine-2-carboxylic acid (A2C) to which all of them proved to be resistant even though this was an unselected trait. The four lines were tested for the intracellular content of some free amino acids. Results of such determination showed that the content of tryptophan and proline was roughly proportional to the degree of resistance of the lines to the two analogues. The fact that all the lines resistant to 5MT over-produced proline suggests that the latter feature may be a direct consequence of the increased pool of free tryptophan. The four cell lines tested showed a rate of tryptophan uptake similar to that of the parental line. On the contrary the rate of proline and A2C by the 5MT-11 cell line was reduced to 23% and 10% of that of the parental line, respectively. (author)

  11. Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.

    Science.gov (United States)

    Ogawa, Yoichi; Dansako, Tomoko; Yano, Kentaro; Sakurai, Nozomu; Suzuki, Hideyuki; Aoki, Koh; Noji, Masaaki; Saito, Kazuki; Shibata, Daisuke

    2008-02-01

    We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

  12. Effects of Polysaccharide Elicitors from Endophytic Fusarium oxysporium Dzf17 on Growth and Diosgenin Production in Cell Suspension Culture of Dioscorea zingiberensis

    Directory of Open Access Journals (Sweden)

    Ligang Zhou

    2011-10-01

    Full Text Available Three polysaccharides, namely exopolysaccharide (EPS, water-extracted mycelial polysaccharide (WPS and sodium hydroxide-extracted mycelial polysaccharide (SPS, were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

  13. Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis.

    Science.gov (United States)

    Li, Peiqin; Mou, Yan; Shan, Tijiang; Xu, Jianmei; Li, Yan; Lu, Shiqiong; Zhou, Ligang

    2011-10-26

    Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

  14. 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.

  15. Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

    Science.gov (United States)

    Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

    2012-03-01

    Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

  16. Production of Betalains by Suspension Cultures of Basella rubra L.

    OpenAIRE

    Okamura, Tokumitsu; Tabuchi, Mikiko; Shinmi, Takako; Matsuo, Shiho; Yuguchi, Aki

    1998-01-01

    The red-violet betacyanins are evidently suitable as natural color additives for some food systems. Red-violet cell suspension cultures of Basella rubra were found to accumulate the betacyanins. The media containing 2, 4-D and kinetin effectively produced red pigments. In particular, in the case of leaf, the medium containing 1μM of 2, 4-D effectively produced red pigments. Betaxanthins was produced better than betacyanins in the case of leaf.

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

    International Nuclear Information System (INIS)

    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)

  18. Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

    OpenAIRE

    Ramani, Shilpa; Jayabaskaran, Chelliah

    2008-01-01

    Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-...

  19. The Structure of Plant Cell Walls: I. The Macromolecular Components of the Walls of Suspension-cultured Sycamore Cells with a Detailed Analysis of the Pectic Polysaccharides.

    Science.gov (United States)

    Talmadge, K W; Keegstra, K; Bauer, W D; Albersheim, P

    1973-01-01

    This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus). These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9% oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16% rhamnogalacturonan, and 21% xyloglucan.The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall.The rhamnogalacturonan consists of an alpha-(1 --> 4)-linked galacturonan chain which is interspersed with 2-linked rhamnosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl- (1 --> 4)-galacturonosyl- (1 --> 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan.The hydroxyprolyl oligo-arabinosides of the hydroxyproline-rich glycoprotein

  20. Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

    OpenAIRE

    Chu, Chia; Lugovtsev, Vladimir; Golding, Hana; Betenbaugh, Michael; Shiloach, Joseph

    2009-01-01

    MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with th...

  1. Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells.

    Science.gov (United States)

    Staehelin, L A; Chapman, R L

    1987-05-01

    Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed

  2. 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

    Three basic chitinases and one basic beta-1,3-glucanase were secreted into the medium when embryogenic cell suspensions of barley (Hordeum vulgare L.) cv. 'Igri' were cultured as undifferentiated aggregates in the presence of 2,4-D. The enzymes were purified by affinity and ion exchange...... 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...

  3. Procyanidins (Condensed Tannins) in Green Cell Suspension Cultures of Douglas Fir Compared with Those in Strawberry and Avocado Leaves by Means of C(18)-Reversed-phase Chromatography.

    Science.gov (United States)

    Stafford, H A; Lester, H H

    1980-12-01

    The procyanidins (the most common type of proanthocyanidin or condensed tannin) from cell suspension cultures derived from cotyledons of Douglas Fir have been compared with those isolated from leaves of strawberry and avocado. Seventy per cent methanol (v/v) extracts from 100 milligrams fresh weight samples were analyzed by a combination of C(18)-reversed-phase columns with high-performance liquid chromatography, and normal phase paper chromatography. (-)-Epicatechin and its oligomers were generally retarded longer on C(18) columns than the corresponding units made of (+)-catechin when eluted with solvents made up of 5% acetic acid alone or mixed with methanol up to 15% (v/v). Douglas fir preparations contained the most complex set of procyanidins and consisted of oligomers of catechin and epicatechin, whereas strawberry and avocado contained mainly (+)-catechin and (-)-epicatechin derivatives, respectively. PMID:16661581

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

    Energy Technology Data Exchange (ETDEWEB)

    Minocha, R.; Shortle, W.C. (USDA Forest Service, Durham (US)); Minocha, S.C.; Long, S.L. (Dept. of Plant Biology, Univ. of New Hamshire, Durham (US))

    1992-01-01

    Increased aluminium (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 polyamines, spermine and spermidine, and their precusor, putrescine, have been implicated in a number of stress responses of plants. Addition of 0.2, 0.5 or 1.0 mM AlCl{sub 3} to cells cultured for 3 days caused a small but significant increase in cellular levels of putrescine at 4 h followed by a sharp decline by 16 h. There was no further decline in levels of putrescine during the next 32 h. Spermidine levels did not change appreciably compared to those in the control cultures. However, spermine levels increased by 2-3-fold at 24 and 48 h. Cellular activities of arginine decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) were both inhibited by 20-25% at 4 and 7 h. Ornithine decarboxylase (ODC; EC 4.1.1.17) was less than 10% of ADC activity at all times. Whereas all concentrations of Al caused a slight decrease in total cell number, cell viability was affected only by 1.0 mM Al. There was a decrease in the cellular levels of Ca, Mg, Na, K, Mn, P and Fe in the cells treated with Al at 4 h, but a significant increase by 16 and 24 h. The results presented here suggest that both the absolute amounts of Al and the length of exposure to it are important for cell toxicity. (au).

  5. Enhancement of phenolics, resveratrol and antioxidant activity by nitrogen enrichment in cell suspension culture of Vitis vinifera.

    Science.gov (United States)

    Sae-Lee, Napaporn; Kerdchoechuen, Orapin; Laohakunjit, Natta

    2014-01-01

    Ammonium nitrate (NH4NO3), an important nitrogen source (34% N), has been used as an elicitor to stimulate plant growth and development as well as induce secondary metabolites under controlled conditions. In the present paper, we investigated the enhancement of cell biomass, total phenolics, resveratrol levels, and antioxidant activity of Vitis vinifera cv. Pok Dum by nitrogen enrichment (MS medium supplemented with NH4NO3 at 0, 500, 1,000, 5,000 and 10,000 mg/L). The highest accumulations of biomass, phenolics and resveratrol contents were observed at 8.8-fold (86.6 g DW/L), 15.9-fold (71.91 mg GAE/g DW) and 5.6-fold (277.89 µg/g DW) by the 14th day, in the medium supplemented with 500 mg/L NH4NO3. Moreover, the antioxidant activities of cultured grape cells estimated by the DPPH· and ABTS·+ assay were positively correlated with phenolics and resveratrol, and the maximum activity was also observed in cultured cells with 500 mg/L NH4NO3 at 176.11 and 267.79 mmol TE/100 g DW, respectively. PMID:24962393

  6. Enhancement of Phenolics, Resveratrol and Antioxidant Activity by Nitrogen Enrichment in Cell Suspension Culture of Vitis vinifera

    Directory of Open Access Journals (Sweden)

    Napaporn Sae-Lee

    2014-06-01

    Full Text Available Ammonium nitrate (NH4NO3, an important nitrogen source (34% N, has been used as an elicitor to stimulate plant growth and development as well as induce secondary metabolites under controlled conditions. In the present paper, we investigated the enhancement of cell biomass, total phenolics, resveratrol levels, and antioxidant activity of Vitis vinifera cv. Pok Dum by nitrogen enrichment (MS medium supplemented with NH4NO3 at 0, 500, 1,000, 5,000 and 10,000 mg/L. The highest accumulations of biomass, phenolics and resveratrol contents were observed at 8.8-fold (86.6 g DW/L, 15.9-fold (71.91 mg GAE/g DW and 5.6-fold (277.89 µg/g DW by the 14th day, in the medium supplemented with 500 mg/L NH4NO3. Moreover, the antioxidant activities of cultured grape cells estimated by the DPPH· and ABTS·+ assay were positively correlated with phenolics and resveratrol, and the maximum activity was also observed in cultured cells with 500 mg/L NH4NO3 at 176.11 and 267.79 mmol TE/100 g DW, respectively.

  7. Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    I. A. Siddiqui

    2013-12-01

    Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

  8. 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. PMID:23661342

  9. 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

    International Nuclear Information System (INIS)

    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

  10. Characterization of cell suspensions from solid tumors

    Energy Technology Data Exchange (ETDEWEB)

    Pallavicini, M.

    1985-07-10

    The desirable features of cells in suspension will necessarily be dependent upon the use for which the cells were prepared. Adequate cell yield or recovery is defined by the measurement to be performed. Retention of cellular morphology is important for microscopic identification of cell types in a heterogenous cell suspension, and may be used to determine whether the cells in suspension are representative of those in the tumor in situ. Different dispersal protocols may yield cells with different degrees of clonogenicity, as well as altered biochemical features, such as loss of cellular proteins, surface antigens, nucleotide pools, etc. The quality of the cell suspension can be judged by the degree of cell clumping and level of cellular debris, both of which impact on flow cytometric measurements and studies in which the number of cells be known accurately. Finally, if the data measured on the cells in suspension are to be extrapolated to phenomena occurring in the tumor in situ, it is desirable that the cells in suspension are representative of those in the solid tumor in vivo. This report compares characteristics of tumor cell suspensions obtained by different types of selected disaggregation methods. 33 refs., 2 figs., 4 tabs.

  11. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures. [Glycine max (L. ); Gossypium hirsutum

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of ({sup 14}C)mevalonate (({sup 14}C)MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, ({sup 14}C)MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with ({sup 14}C)clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  12. Somatic embryogenesis in Picea suspension cultures.

    Science.gov (United States)

    Stasolla, Claudio

    2006-01-01

    Generation of somatic embryos in spruce is achieved through the execution of five steps designated as: (1) induction of embryogenic tissue, (2) maintenance of embryogenic tissue, (3) embryo development, (4) embryo maturation, and (5) conversion into plants. Depending on species and genotypes within the same species, each step must be optimized for obtaining maximum results. In general, embryogenic tissue is generated from immature and mature zygotic embryos and maintained in either liquid or solid conditions in the presence of plant growth regulators auxin and cytokinin. Initiation of embryo development in suspension cultured is induced by removal of plant growth regulators, whereas continuation of development and completion of maturation require applications of abscisic acid and imposition of a desiccation period. Both treatments are needed for conferring morphological and physiological maturation to the embryos. Mature somatic embryos are germinated in the absence of plant regulators and embryo conversion (i.e., formation of a functional shoot and root, occurs after a few weeks in culture). PMID:16673908

  13. An Optimized Method for Suspension Culture of CHO Cells to Produce Recombinant Human Erythropoietin (EPO)%悬浮培养CHO细胞生产重组人促红细胞生成素条件的优化

    Institute of Scientific and Technical Information of China (English)

    杨栋; 牛红军; 陆刚; 史嘉林; 孙浩明; 李晖

    2012-01-01

    Objective: To screen and domesticate the adherent cultured CHO cells to obtain high expression of cell suspension culture for production of recombinant human erythropoietin erythropoietin (rHuEPO). Methods: Using 96-well and 24-well plates culture method to screen and domesticate the highly expressing CHO cell strain. Acclimate the high expression cell strain and make it suitable for suspension culture. It's inoculated into the bioreactor in serum-free culture after amplified by the shake flask, and monitoring of glucose content, measuring rHuEPO expression of daily. Results: The suspension culture of CHO cell production of rHuEPO has short production period, higher expression than adherent culture. On the other hand, it is easy to operate and scale-up, but not easy to pollute. Furthermore, we established of the CHO cell strain for suspension culture,which provided a technical basis for industrial production of CHO cells the rHuEPO. Conclusion: After process optimization, the use of serum-free suspension culture production of erythropoietin average expression has high, short production period, low cost of production.than adherent culture.%目的:通过对贴壁培养CHO细胞筛选驯化,得到高表达的细胞后进行悬浮培养生产重组人促红细胞生成素(rHuEPO).方法:利用96孔板和24孔板对CHO细胞进行筛选,得到高表达细胞株后进行驯化,使其适合悬浮培养,经过摇瓶扩增后接种到生物反应器中无血清培养,每天监测葡萄糖含量,测rHuEPO表达量.结果:悬浮培养CHO细胞生产rHuEPO,生产周期短,表达量比贴壁培养高出很多,操作方便,减少污染,易于放大,并建立了适合悬浮培养的CHO细胞株,为工业化悬浮培养CHO细胞生产rHuEPO提供了技术基础.结论:经过工艺优化后利用无血清悬浮培养生产促红细胞生成素平均表达量较贴壁培养高,生产周期短,有利于降低生产成本.

  14. THE CHANGE OF KINETIK PARAMETERS OF THE WEAK-ASSOCIATED WITH WALL CELL PEROXIDASE IN THE SUSPENSION CULTURE OF POTATO CELLS IN THE BEGINNING OF INFECTION

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2006-03-01

    Full Text Available The change in kinetic parameters of extracellular peroxidase of suspension cells of resistant potato variety (Lugovskoi and sensitive variety (Luk,ynovskii in the initial period of infection by 5369 Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. pathogen was examined. Extracellular peroxidases of resistant and sensitive potato variety without pathogens were shown to be concurrently inhibited. At the beginning of infection enzyme activity was extremely increased due to enhanced affinity to substrate as a result of reducing of competitive inhibiting. In increasing enzyme activity is sensitive potato variant evidently caused by other mechanism.

  15. The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris).

    Science.gov (United States)

    Wilder, B M; Albersheim, P

    1973-05-01

    The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.The present paper describes the structure of a xyloglucan present in the walls and in the extracellular medium of suspension-cultured Red Kidney bean (Phaseolus vulgaris) cells and compares the structure of the bean xyloglucan with the structure of the sycamore xyloglucan. Although some minor differences were found, the basic structure of the xyloglucans in the cell walls of these distantly related species is the same. The structure is based on a repeating heptasaccharide unit which consists of four residues of beta-1, 4-linked glucose and three residues of terminal xylose linked to the 6 position of three of the glucosyl residues.

  16. 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. PMID:24589476

  17. 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.

  18. Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

    OpenAIRE

    Wagner, V T; Matthysse, A G

    1992-01-01

    Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or...

  19. Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

    Science.gov (United States)

    Ramani, Shilpa; Jayabaskaran, Chelliah

    2008-01-01

    Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation. PMID:18439256

  20. 川贝母细胞团块悬浮培养生产生物碱的研究%Study on the Production of Alkaloid by Cell Mass Suspension Culture of Fritillaria cirrhosa

    Institute of Scientific and Technical Information of China (English)

    王跃华; 何宗晟; 孙雁霞; 马良良; 刘益丽; 林抗雪

    2011-01-01

    目的:建立川贝母细胞团块悬浮培养体系,筛选出快速获取细胞团块增殖的最佳培养条件和激素配比.方法:以MS培养基为基本培养基,比较了接种量、激素配比和生长调节物质等对川贝母细胞团块悬浮培养的影响,并比较了细胞团块在不同培养条件下的生长情况,测定了增殖细胞团块中总生物碱含量.结果与结论:用川贝母细胞团块进行悬浮培养,其生长速度明显快于固体培养;细胞团块中总生物碱含量均高于市售川贝母和野生川贝母鳞茎;川贝母细胞团块悬浮培养最适的接种量为30g/L,最佳培养基为MS+6-BA2.0 mg/L+NAA 0.2 mg/L.%Objective:Set up Fritillaria cirrhosa cell mass suspension culture system to rapidly screen the best culture conditions for cell mass proliferation and hormone combination. Methods: Using MS medium as the basic medium, the impact of inoculum size, hormone combination,growth regulators for Fritillaria cirrhosa cell mass suspension culture were compared,and also the growth of cell mass at different culture conditions was compared,and the total alkaloids content in proliferative cell mass was measured. Results and Conclusion: Fritillaria cirrhosa grow significantly faster in cell mass suspension culture than in the solid culture. The total alkaloid content in cell mass is higher than commercial and wild bulb of Fritillaria cirrhosa. The optimal inoculum size for cell mass suspension culture is 30 g/L and the optimal culture media is MS + 6-BA 2.0 mg/L + NAA 0.2 mg/L

  1. Evaluation of nephrotoxicity in vitro using a suspension of highly purified porcine proximal tubular cells and characterization of the cells in primary culture.

    Science.gov (United States)

    Kruidering, M; Maasdam, D H; Prins, F A; de Heer, E; Mulder, G J; Nagelkerke, J F

    1994-01-01

    Proximal tubular cells (PTC) were isolated from porcine kidney by collagenase treatment, subsequently purified on a discontinuous density gradient and finally cultured. Porcine PTC (PPTC) in primary culture expressed keratin, characteristics of epithelia and brush border specific glycoproteins (FX1A). In addition, vimentin was present. All cells were negative for the endothelial marker pal-E. Less than 0.1% expressed the Tamm-Horsfall protein, characteristic of the distal tubule, while less than 0.3% of all cells in culture expressed desmin, characteristic of connective tissue (i.e. fibroblasts) and mesangial cells. Ultrastructural analysis revealed microvilli, tight junctions and abundant mitochondrial and lysosomes, all characteristics of proximal tubular cells. Freshly isolated PPTC were validated as in vitro model to detect nephrotoxicity by studying the effect of mercuric chloride, cis-platin, p-aminophenol and the halogenated alkenes 1,2 dichlorovinyl-l-cysteine, S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine (DCDFE-cys) and the glutathione conjugate of DCDFE on viability and mitochondrial membrane potential. The cells responded, time- and dose-dependently, to the nephrotoxic compounds with a decrease in mitochondrial membrane potential and loss of viability. The sensitivity of the porcine cells in detecting toxic effects corresponded favorably with in vitro systems derived from other animals. PMID:7859034

  2. Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ali, M S; Akazawa, T

    1986-05-01

    The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F(0) to F(1) ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N'-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.

  3. 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.)

  4. Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota) 1

    Science.gov (United States)

    Ohyama, K.; Gamborg, O. L.; Miller, R. A.

    1972-01-01

    A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation. The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 × 108, and for carrot DNA it was 1.56 × 108. Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter. PMID:16658166

  5. Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota).

    Science.gov (United States)

    Ohyama, K; Gamborg, O L; Miller, R A

    1972-09-01

    A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation.The melting temperature of ammi and carrot DNA in 0.15 m NaCl and 15 mm trisodium citrate buffer, pH 7.0, was 84.0 C and 84.5 C, respectively. The molecular weight for ammi DNA was 1.43 x 10(8), and for carrot DNA it was 1.56 x 10(8). Ammi DNA exhibited a single band at 1.690 grams per cubic centimeter in CsCl, whereas carrot DNA showed two bands, one at 1.693 grams per cubic centimeter and another at 1.706 grams per cubic centimeter. Ammi DNA consisted of a doublestranded form, since denaturation of the DNA caused a complete upward shift of 0.020 grams per cubic centimeter.

  6. Studies on Genetic Transformatiom of Embryogenic Suspension Cultures of Sweetpotato

    Institute of Scientific and Technical Information of China (English)

    ZHAI Hong; LIU Qing-chang

    2003-01-01

    Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang wasconducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase (GUS) and neomycin phosphotransferase (NPT Ⅱ ) genes. The results indicated that embryogenicsuspension cultures precultured for 1 -3 d were suitable for the transformation. The optimal cocultivation timewas 4 - 5 d. The optimal concentration of kanamycin was 50-75 mg L-1 for suspension culture and 100 mg L-1for embryogenic callus proliferation and plant regeneration. The optimal concentration of carbencillin was 100mg L-1. Transgenic plants identified with GUS assays and PCR analyses were obtained.

  7. Visnagin: biosynthesis and isolation from Ammi visnagi suspension cultures.

    Science.gov (United States)

    Kaul, B; Staba, E J

    1965-12-24

    During an examination of Ammi visnaga Lam. suspension cultures for the biosynthesis of furanochromones and related medicinal compounds, visnagin was isolated in crystalline form and identified. Thus, certain medicinally important secondary plant metabolites may be produced in appreciable amounts by plant tissue cultures.

  8. Techniques for mammalian cell tissue culture.

    Science.gov (United States)

    Phelan, Mary C

    2006-05-01

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

  9. [Cell cultures].

    Science.gov (United States)

    Cipro, Simon; Groh, Tomáš

    2014-01-01

    Cell or tissue cultures (both terms are interchangeable) represent a complex process by which eukaryotic cells are maintained in vitro outside their natural environment. They have a broad usage covering not only scientific field but also diagnostic one since they represent the most important way of monoclonal antibodies production which are used for both diagnostic and therapeutic purposes. Cell cultures are also used as a "cultivation medium" in virology and for establishing proliferating cells in cytodiagnostics. They are well-established and easy-to-handle models in the area of research, e.g. as a precious source of nucleic acids or proteins. This paper briefly summarizes their importance and methods as well as the pitfalls of the cultivation and new trends in this field. PMID:24624984

  10. The rules on suspension cell batch culture of Hedyotis diffusa%白花蛇舌草悬浮细胞分批培养规律的研究

    Institute of Scientific and Technical Information of China (English)

    于瑞莲; 许金国; 顾晓娟

    2012-01-01

    目的 利用白花蛇舌草茎尖的分生组织建立植物悬浮细胞培养系,确定白花蛇舌草悬浮细胞分批培养时的变化规律.方法 通过接种不同量的细胞液确定最适接种量,以细胞干重、蔗糖、铵离子、硝酸盐氮和多糖含量作为检测指标,确定白花蛇舌草悬浮细胞分批培养时的变化规律.结果 白花蛇舌草悬浮细胞分批培养的最适接种量为15%,此时细胞干重达到最大值.在此接种量下,当培养时间达到9d时,pH很快降低到3左右,细胞干重不再增加.培养液中的主要营养成分碳源——蔗糖,在培养开始时,有比较大的降低,在培养后期,细胞干重不再增加时,也不再有大的改变.氮源[N03-]优于[NH4+]先被利用,而且[N0f]的利用速率要远高于[NH4+],达到7.14 μg/(mL·d);细胞液中多糖的生成和细胞的生长属于非偶联型,在培养后期,逐渐大量生成.结论 初步确定白花蛇舌草悬浮细胞分批培养时的变化规律,为以后的培养工艺优化打下了基础.%Purpose Hedyotis diffusa suspension cell was established by the tip of meristem to find out the rules on suspension cell batch culture of Hedyotis diffusa with the time. Methods The optimum inoculum was determined by different inoculation of cell medium. The rules on suspension cell batch culture of Hedyotis diffusa with the time were found by the detection of the amount of cell dry weight, sucrose, ammonium, nitrate and polysaccharide. Results The suspension cell batch culture of Hedyotis diffusa optimum inoculum is 15% .maximum dry cell weight at the same time. At this inoculation,when the incubation time was of 9 days,and pH quickly reduced to about 3 ,the cell dry weight did not increase. The main medium nutrients were carbon -sucrose, a relatively large decreased in the beginning of culture, and the cell dry weight no longer had a big change in the late culture stage. Nitrogen [ NO3-] was used prior to [ NH4+ ] ,and the use

  11. Isolation and culture of suspension protoplasts of vetiver

    Directory of Open Access Journals (Sweden)

    Somporn Prasertsongskun

    2005-05-01

    Full Text Available In this research, protoplasts were isolated from cell suspension derived from inflorescence of vetiver (Vetiveria zizanioides Nash Surat Thani germplasm. The optimum condition for protoplast isolation was established by using 2% cellulase Onozuka R10, 2% macerozyme R10, 0.5% pectinase in 0.4 M mannitol and 7 mM CaCl2.2H2O at pH 5.8 and incubated for 10 hours in the dark on the rotary shaker at 50 rpm. Maximum protoplast yields were 8.4 × 104 protoplasts/ml PCV. Division of protoplasts was observed only in liquid medium. The first cell division was observed after 3 days of culture initiation, and the average division was 5.0% in the N6 medium supplemented with 1.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid and 0.5 mg/l BA (Benzyladenine. An optimal density for culture division was 1 × 105 protoplasts/ml.

  12. 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

  13. Surfactant-induced non-lethal release of anthraquinones from suspension culture of Morinda citrifolia.

    NARCIS (Netherlands)

    Bassetti, L.; Hagendoorn, M.J.M.; Tramper, J.

    1995-01-01

    A new approach based on the use of the surfactant Pluronic F-68 to obtain non-lethal release of plant cell intracellular products was investigated. Suspension cultures of Morinda citrifolia (Rubiaceae), producing anthraquinones as secondary metabolites, were selected as model system. By supplementin

  14. 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

  15. DNA analysis of epithelial cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  16. Biotransformation of artemisinin by Catharanthus roseus and Ginkgo biloba cell suspension cultures%长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究

    Institute of Scientific and Technical Information of China (English)

    韩健; 戴均贵; 崔亚君; 占纪勋; 郭洪祝; 果德安

    2003-01-01

    Object To investigate the biotransformation of the antimalarial compound artemisinin( Ⅰ ) by Catharanthus roseus and Ginkgo biloba cell suspension cultures. Methods Plant tissue culture technology was employed. The product was isolated on silica gel column chromatography and its structure was elucidated by spectroscopic evidence. Results One product was obtained and its structure was characterized as 3α- hydroxydeoxyartemisinin ( Ⅱ ). Conclusion Both of C. roseus and G. biloba cell suspension cultures can bioconvert artemisinin.%目的对抗疟药物青蒿素(Ⅰ)进行了生物转化研究.方法利用长春花及银杏植物细胞悬浮培养细胞进行生物转化.用硅胶柱色谱进行产物的分离,波谱方法鉴定产物的结构.结果此两种植物悬浮细胞体系均能将青蒿素转化成3α-羟基去氧青蒿素(Ⅱ).结论此两种植物悬浮细胞体系均能有效转化青蒿素.

  17. Phytophthora elicitor PB90 induced apoptosis in suspension cultures of tobacco

    Institute of Scientific and Technical Information of China (English)

    JI Rui; ZHANG Zhengguang; WANG Yuanchao; ZHENG Xiaobo

    2005-01-01

    The protein elicitor PB90 secreted by Phytophthora boehmeriae is an efficient elicitor inducing the hypersensitive response and systemic acquired resistance in tobacco plants. Here, we observed cell death in suspension-cultured cells of Nicotiana tabacum BY-2 with PB90 treatment using Trypan blue staining method. And this cell death could be suppressed by cycloheximide, an inhibitor of proteins synthesis, which implies that PB90-induced cell death was an active cell death process requiring new protein synthesis. DAPI staining revealed that PB90 induce rapid chromatin condensation, margination, apoptotic bodies' formation and DNA laddering, further TUNEL assay also observed the specific breakage of 3′-OH ends. All of the above common morphological characteristics indicated that PB90 induced apoptosis in suspension cultures of tobacco, suggesting that hypersensitive response induced by PB90 is an apoptotic process.

  18. 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

  19. 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

  20. Beryllium toxicity testing in the suspension culture of mouse fibroblasts.

    Science.gov (United States)

    Rössner, P; Bencko, V

    1980-01-01

    Suspension culture of mouse fibroblast cell line L-A 115 was used to test beryllium toxicity in the presence of magnesium ions. Beryllium added to the MEM cultivation medium was bound in a complex with sulphosalicylic acid BeSSA complex, because the use of beryllium chloride turned out to yield ineffective beryllium phosphate that formed macroscopically detectable insoluble opacities. The BeSSA complex was used in the concentration range: 10(-3)--10(-9)M, magnesium was used in 3 concentrations: 10(-1)M, 5 x 10(-2)M and 10(-2)M. Growth curve analysis revealed pronounced beryllium toxicity at the concentration of 10(-3)M, magnesium-produced toxic changes were observed only at the concentration of 10(-1)M. No competition between the beryllium and magnesium ions was recorded. It is assumed that the possible beryllium-magnesium competition was significantly modified by the use of BeSSA complex-bound beryllium.

  1. Cell suspension culture and plant regeneration of a Brazilian plantain, cultivar Terra Cultura de células em suspensão e regeneração de plantas de bananeira cultivar Terra

    Directory of Open Access Journals (Sweden)

    Lucymeire Souza Morais-Lino

    2008-10-01

    Full Text Available The objective of this study was to establish cell suspension culture and plant regeneration via somatic embryogenesis of a Brazilian plantain, cultivar Terra Maranhão, AAB. Immature male flowers were used as explant source for generating highly embryogenic cultures 45 days after inoculation, which were used for establishment of cell suspension culture and multiplication of secondary somatic embryos. Five semisolid culture media were tested for differentiation, maturation, somatic embryos germination and for plant regeneration. An average of 558 plants per one milliliter of 5% SCV (settled cell volume were regenerated in the MS medium, with 11.4 µM indolacetic acid and 2.2 µM 6-benzylaminopurine. Regenerated plants showed a normal development, and no visible somaclonal variation was observed in vitro. It is possible to regenerate plants from cell suspensions of plantain banana cultivar Terra using MS medium supplemented with 11.4 µM of IAA and 2.2 µM of BAP.O objetivo deste trabalho foi estabelecer a cultura de células em suspensão e a regeneração de plantas via embriogênese somática de bananeira cultivar Terra Maranhão, AAB. Flores imaturas masculinas foram utilizadas como fonte de explante para obtenção de culturas altamente embriogênicas 45 dias após a inoculação, as quais foram utilizadas para o estabelecimento de suspensões celulares e multiplicação de embriões somáticos secundários. Cinco meios de cultura semi-sólidos foram testados para a diferenciação, maturação, germinação dos embriões somáticos e para a regeneração de plantas. A média de 558 plantas por mililitro de 5% SCV (volume de células sedimentadas foi regenerada, em meio MS com 11,4 µM de ácido indolacético e 2,2 µM de 6-benzilaminopurina. As plantas regeneradas apresentaram desenvolvimento normal, e não foi observada a ocorrência de variação somaclonal in vitro. É possível a regeneração de plantas a partir de células em suspens

  2. Efficient amplification of chimeric adenovirus 5/40S vectors carrying the short fiber protein of Ad40 in suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Marta Miralles

    Full Text Available The human adenovirus 40 (Ad40 is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.

  3. Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension.

    Science.gov (United States)

    Monteil, Dominique T; Shen, Xiao; Tontodonati, Giulia; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-07-15

    Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension. PMID:27130502

  4. Comparison of the oxygen exchange between photosynthetic cell suspensions and detached leaves of Euphorbia characias L

    Energy Technology Data Exchange (ETDEWEB)

    Carrier, P.; Chagvardieff, P.; Tapie, P. (C.E.N. Cadarache, Saint-Paul lez Durance (France))

    1989-11-01

    Using a mass-spectrometric {sup 16}O{sub 2}/{sup 18}O{sub 2}-isotope technique, we compared the nature and the relative importance of oxygen exchange in photomixotrophic (PM) and photoautotrophic (PA) suspensions of Euphorbia characias L. with those in intact leaves of the same species. Young and mature leaves, dividing and nondividing cell suspensions were characterized in short-term experiments. On chlorophyll basis, the gross photosynthetic activities at CO{sub 2} saturating concentration of PA and PM suspensions varied little from those of leaves. On dry weight basis, gross photosynthesis of PA suspensions was equal to that of leaves because of their similar chlorophyll content. This was not the case in PM suspensions where gross photosynthesis was lower and largely varied during the growth cycle. The CO{sub 2} compensation point of PA cells was much higher than that of leaves. Oxygen uptakes were analyzed in terms of mitochondrial respiration, photorespiration and light stimulation of oxygen uptake (LSOU), often identified to Mehler-type reactions. In Pa and PM suspensions, mitochondrial respiration rates were higher than in leaves by a factor of 1.5 to 4.5. In PM suspensions, photorespiration and LSOU were observed only in nondividing cells. Photorespiration and LSOU rates were comparable in PA suspensions and leaves. Our results demonstrate that photorespiration of PA suspensions has not been affected by the 2% CO{sub 2} concentration imposed during 2 years of culture.

  5. Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures.

    Science.gov (United States)

    Leone, A.; Costa, A.; Tucci, M.; Grillo, S.

    1994-10-01

    To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)- mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged

  6. The influence of fast neutron irradiation through micropropagation, calli induction and cell aggregate suspension culture of tapak dara cultivate vinca rosea linn

    International Nuclear Information System (INIS)

    Study on the influence of fast neutron irradiation toward tissue induction of apical shoot, calli of leaf and corolla as well as development of bud micropropagation using variety of MS and Gamborg (B5) which were supplemented with growth hormone 2,4-D NAA, BAP and kinetin has been carried out. Cell aggregates were obtained from modified liquid media by mixing MS macro element and Gamborg vitamin. Influence of the iow level irradiation (0,5-10 Gy) was investigated for auxiliary bud micropropagation , middle (5 - 20 Gy) for calli induction, while for call aggregates higher doses (until 30 Gy) were used. Optimum growth of bud micropropagation was stimulate at dose range between 0,5-1Gy and grown on MS supplemented whit BAP and NAAN, while for leaf and corolla calli was at 5 Gy, on MS media which was supplemented whit 1 mg/L kinetin, 10mg/L BAP and 0,5 mg/L NAA. However, neutron dose of 10 Gy decreased the induction of leaf and corolla calli. The highest radioresistance was shown by cell aggregates of leaf calli that grew prosperously up to 20Gy. Key words : fast neutron, micropropagation, tissue culture, cell culture, vinca rosea L

  7. Automated single cell isolation from suspension with computer vision.

    Science.gov (United States)

    Ungai-Salánki, Rita; Gerecsei, Tamás; Fürjes, Péter; Orgovan, Norbert; Sándor, Noémi; Holczer, Eszter; Horvath, Robert; Szabó, Bálint

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1-2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved. PMID:26856740

  8. 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.

  9. Cellular aggregation is a key parameter associated with long term variability in paclitaxel accumulation in Taxus suspension cultures

    OpenAIRE

    Patil, Rohan A.; Kolewe, Martin E.; Roberts, Susan C.

    2012-01-01

    Plant cell cultures provide a renewable source for synthesis and supply of commercially valuable plant-derived products, particularly for secondary metabolites. However, instability in product yields over multiple passages has hampered the efficient and sustainable use of this technology. Paclitaxel accumulation in Taxus cell suspension culture was quantified over multiple passages and correlated to mean aggregate size, extracellular sugar level, ploidy, and cell cycle distribution. Paclitaxe...

  10. Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures.

    Science.gov (United States)

    Kim, Beum Jun; Gibson, Donna M; Shuler, Michael L

    2004-01-01

    The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.

  11. Metabolism of Aromatic Amino Acids during the Growth Cycle of Batch Suspension Cultures of Catharanthus roseus

    OpenAIRE

    Nagaoka, Noriko; ASHIHARA, Hiroshi

    1988-01-01

    Profiles of the levels and metabolism of aromatic compounds in suspension-cultured cells of Catharanthus roseus during the growth cycle were determined. The level of total protein-amino acids, i.e., sum of the amounts of amino acids in hydrolyzates of proteins, and the level of total phenolic acids increased after transfer of the cells in the stationary phase to fresh Murashige-Skoog medium. The maximum levels of the proteinamino acids and those of the phenolic acids were observed on days 3-5...

  12. NO-mediated hypersensitive responses of rice suspension cultures induced by incompatible elicitor

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Magnaporthe grisea special races 98-186-1G1 and 97-23-2D1 induce incompatible and compatible reactions respectively with rice "Xiushui". The elicitor from the cell wall of M. grisea race 98-186-1G1, termed IE, strongly induced theHR response in suspension cultures of rice "Xiushui", including increased PAL activity, transcription of pal , pr1, chi, cell death and the generation of NO. The elicitor prepared from the cell wall of M. grisea race 97-23-2D1, named CE, was much less efficient at inducing such effects. The NOS enzyme inhibitors L-NAAand PBITU suppressed the production of NO induced by IE in "Xiushui" rice. The increased PAL activity and transcription of pr1, pal, chi genes induced by IE were blocked by L-NAA, PBITU or CPTIO pretreatment. Direct treatment of rice cultures with the NO donor (SNP) also induced the transcription of pr1, pal and chi genes. These data implicated that NO acted as a signal mediating the HR induced by IE in rice and showed that NO, in combination with H2O2, is necessary for induction of cell death by IE in rice suspension cells.

  13. 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.

  14. 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

    and fluorochrome labelling of resin-embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls...... in habituated cells also diminished with the increasing number of subcultures. Habituated cells also liberated less extensin into the medium. In habituated cells, a decrease in the cell wall arabinogalactan protein (AGP) labelling was observed both in cell walls and in the culture medium. The increase...... in the number of subcultures in 0.3 µM dichlobenil was accompanied by an increment in some pectic epitopes (JIM5 and LM5) and a decrease in other pectic and in protein epitopes (JIM7, PAM1, LM6, LM2 and MAC207), indicating a re-structuring of cell walls throughout the habituation procedure. Dehabituated cells...

  15. 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

  16. 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...

  17. Enrichment of Breast Cancer Stem Cells by Serum-free Shaking Suspension Culture%无血清摇动悬浮培养富集乳腺癌干细胞

    Institute of Scientific and Technical Information of China (English)

    闫文星; 陈玉丙; 张红梅; 国春龙; 王铁君

    2011-01-01

    Objective To investigate the characteristics of division of breast cancer MCF-7 cells under various conditions so as to develop a rapid and effective method for enrichment of breast cancer stem cells. Methods MCF-7 cells were subjected to static culture in complete medium (group A), shaking culture in complete medium (group B), static culture with cytokine (group C) and shaking culture with cytokine (group D) respectively, for 12, 24 and 36 h, then observed for division under inverted phase contrast microscope, based on which clone formation rate was calculated, and determined for the percentage of CD44+/CD24"/low lymphocyte subsets. Results In groups B and C, rod-like divisions were observed in both about 30% of cells 12 h, and in about 50% and about 60% of cells respectively 24 h, while large cell clones were formed 36 h after culture. However, in group D, rod-like division was observed in about 50% of cells 12 h, and in about 80% of cells 24 h when several cell clones appeared, while the number of cell clones decreased 36 h after culture. The percentage of CD44+/CD24"/km lymphocyte subsets 12 h after culture in group D (8. 05%) was 8 times of those in group B (0. 99%) and 2. 1 times of those in group C (3. 80%). However, the percentage 24 h after culture in group D (15. 24%) was 3 times of those in group B (4. 83%) and 6 times of those in group C (2. 30%). The percentage in group D decreased to 9. 68% 36 h after culture, which was about 9 times of those in groups B (0. 95%) and C (1. 03%). Conclusion The mitotic division of MCF-7 cells was accelerated in shaking culture with cytokine, while the percentage of CD44+/CD24'/low lymphocyte subsets increased rapidly, and stem cell pool increased significantly, indicating serum-free shaking suspension culture a rapid and effective method for enrichment of breast cancer stem cells.%目的 探讨不同培养条件下乳腺癌MCF-7细胞的分裂特点,建立快速、有效的乳腺癌干细胞富集方法.方法 将MCF-7

  18. Fish Stem Cell Cultures

    OpenAIRE

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  19. 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...... to the MS suspension cultures. Sucrose was hydrolysed into its monosaccharide components in the culture medium. Glucose was depleted faster than fructose indicating a preference for glucose as a carbohydrate source of the M. ogiformis cultures. The high water uptake by the suspension aggregates 12 to 15...

  20. 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

    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...

  1. Cell Culture for Production of Insecticidal Viruses.

    Science.gov (United States)

    Reid, Steven; Chan, Leslie C L; Matindoost, Leila; Pushparajan, Charlotte; Visnovsky, Gabriel

    2016-01-01

    While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line. PMID:27565495

  2. Optimization and influencing factor of electroporation parameters for cells cultured in suspension%悬浮细胞电转染条件的优化及影响因素

    Institute of Scientific and Technical Information of China (English)

    龙潺; 唐雪元

    2008-01-01

    Objective To optimize the electroporation parameters in leukemia cell lines cultured in suspension using green fluorescent protein(GFP)as a reporter gene.Methods The GFP plasmid was transferred into leukemia cell lines K562 by electroporation using differently experimental conditions such as the voltage(200-400 V),the electric capacity(450-1200μF),the state of cell vitality and the serum concentration with buffer solution(0,10%,15%).The electroporation efficiency was evaluated bv flow cytometry and fluorescent microscopy.Results The highest electropration efficiency with leukemia cell lines k562 cultured in suspension was obtained under the condition of voltage 3 10 V,electric eapacity of 1050μF(for pEGFP-C2/K562,67.04%;for pEGFP-C2/BRD7/K562,59.29%).The electmporation efficieney for the cells in logarithmic growth phase Was increaSed highly.The seruln concentration in buffer solution was not related with the electropration efficiency.Conclusion Electroporation is a method with high efficiency to gene transfeetion,and optimizing eleetroporation parameters and controlling the related factors can increase the eleetmporation efficiency.%目的 以绿色荧光蛋白(GFP)为报告基因,优化悬浮培养的白血病细胞的电穿孔转染条件.方法 通过控制电压(200~400 V)、电容(450~1200μF)、细胞状态及缓冲液血清浓度(0%、10%、15%)等转染条件,采用不同条件组合后用电穿孔法将质粒转入悬浮培养的人白血病细胞株K562,通过流式细胞仪和荧光显微镜分析转染率.结果 K562细胞在310 V、1050μF条件下转染率最高,pEGFP-C2/K562为67.04%,pEGFP-C2/BRD7/K562为59.29%.对数生长期细胞电转染率高于生长过老期细胞;而缓冲液中的血清浓度与电转染率无关.结论 电穿孔是一种高效的基因转染法,通过优化转染条件、控制影响因素,可提高转染率.

  3. Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells.

    Science.gov (United States)

    Driouich, A; Zhang, G F; Staehelin, L A

    1993-04-01

    Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [3H]xylose and

  4. Production of Arbutin through Biotransformation of Exogenous Hydroquinone by Datura stramonium Cell Suspension Cultures%白花曼陀罗细胞悬浮培养生物转化外源氢醌合成熊果苷的研究

    Institute of Scientific and Technical Information of China (English)

    彭春秀; 龚加顺

    2006-01-01

    研究了白花曼陀罗细胞悬浮培养对外源氢醌的糖基化.转化细胞来自白花曼陀罗嫩茎在LS固体培养基上诱导产生的愈伤组织.白花曼陀罗悬浮培养细胞不能分泌熊果苷,但能糖基化外源氢醌合成熊果苷.当氢醌添加量达240 μmol/100mL培养物时,约有93.4%的氢醌转化形成了熊果苷,并应用多种色谱技术进行分离纯化,进行了HPLC分析和结构鉴定.%To investigate the biotransformation of hydroquinone by cell suspension cultures of Datura stramonium. Cultured cells derived from stems of Datura stramonium were maintained in Linsmaiher and Skoog (LS) solid medium. Datura stramonium cells in suspension cultures did not accumulate arbutin (4-hydroxyphenyl-β-D-glucopyranoside) but were able to specifically o-glucosylate exogenous hydroquinone at position 1. In particular, Datura cultures glucosylated ca 93.4% of hydroquinone (240 μmol/100 mL cultures) within 8 days after hydroquinone administered. The arbutin obtained was extracted from the cultures and further purified by silicon Gel column chromatography. The exogenous hydroquinone and arbutin were analyzed by HPLC.

  5. 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.

  6. Improved oxidative tolerance in suspension-cultured cells of C4-pepctransgenic rice by H2O2 and Ca2+under PEG-6000

    Institute of Scientific and Technical Information of China (English)

    Baoyun Qian; Xia Li; Xiaolong Liu; Man Wang

    2015-01-01

    To understand the molecular responses of PC (Overexpressing the maize C4‐pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC)), to drought stress at cel level, we analyzed changes in the levels of signaling molecules (hydrogen peroxide (H2O2), calcium ion (Ca2þ), and nitric oxide (NO)) in suspension‐cultured PC and wild‐type (WT) rice (Oryza sativa L.) cel under drought stress induced by 20%polyethylene glycol 6000 (PEG‐6000). Results demonstrated that PC improved drought tolerance by enhancing antioxidant defense, retaining higher relative water content, survival percentages, and dry weight of cel s. In addition, PEPC activity in PC under PEG treatment was strengthened by addition of H2O2 inhibitor, dimethylthiourea (DMTU) and NO synthesis inhibitor, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), respectively, while that in PC was weakened by addition of free calcium chelator, ethylene glycol‐bis(b‐aminoethylether)‐N,N,N0 ,N0‐tetraacetic acid (EGTA) þ calcium channel outflow inhibitor, ruthenium red (RR) þ plasma membrane channel blocker La(NO3)3, but EGTA þ RR did not. Results also showed that NO and Ca2þ was lying downstream of H2O2 in drought‐induced signaling. Calcium ion was also involved in the expression of C4‐pepc in PC. These results suggested that PC could improve oxidative tolerance in suspension‐cultured cel s and the acquisition of this tolerance required downregulation of H2O2 and the entry of extracel ular Ca2þ into cel s across the plasma membrane for regulation of PEPC activity and C4‐pepc expression.

  7. Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms

    Directory of Open Access Journals (Sweden)

    Jaime Aparecido Cury

    2008-09-01

    Full Text Available The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W, suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0 and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg of crude preparation of RNA per 100 mg of total cell (or biofilm dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg of purified RNA per 100 mg of total cell (or biofilm dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.

  8. Suspensions

    Science.gov (United States)

    Braccini, Stefano

    2000-06-01

    Special suspension systems are used in gravitational wave detectors to reduce the transmission of seismic vibrations to test masses by many orders of magnitude. In ground-based interferometric antennas, this allows to detect gravitational signals even below a few tens of Hz, where seismic vibrations are very strong. The state of the art on this topic is presented. .

  9. Optimizing stem cell culture.

    Science.gov (United States)

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  10. Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

    OpenAIRE

    Hoshino, Yoichiro; Kashihara, Yukiko; Hirano, Tomonari; MURATA, Naho; Shinoda, Koichi

    2008-01-01

    Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 d after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloro...

  11. Proteomic Analysis of a Poplar Cell Suspension Culture Suggests a Major Role of Protein S-Acylation in Diverse Cellular Processes.

    Science.gov (United States)

    Srivastava, Vaibhav; Weber, Joseph R; Malm, Erik; Fouke, Bruce W; Bulone, Vincent

    2016-01-01

    S-acylation is a reversible post-translational modification of proteins known to be involved in membrane targeting, subcellular trafficking, and the determination of a great variety of functional properties of proteins. The aim of this work was to identify S-acylated proteins in poplar. The use of an acyl-biotin exchange method and mass spectrometry allowed the identification of around 450 S-acylated proteins, which were subdivided into three major groups of proteins involved in transport, signal transduction, and response to stress, respectively. The largest group of S-acylated proteins was the protein kinase superfamily. Soluble N-ethylmaleimide-sensitive factor-activating protein receptors, band 7 family proteins and tetraspanins, all primarily related to intracellular trafficking, were also identified. In addition, cell wall related proteins, including cellulose synthases and other glucan synthases, were found to be S-acylated. Twenty four of the identified S-acylated proteins were also enriched in detergent-resistant membrane microdomains, suggesting S-acylation plays a key role in the localization of proteins to specialized plasma membrane subdomains. This dataset promises to enhance our current understanding of the various functions of S-acylated proteins in plants. PMID:27148305

  12. [Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera].

    Science.gov (United States)

    Qu, Jun-Ge; Zhang, Wei; Hu, Quan-Li; Jin, Mei-Fang

    2006-11-01

    The commercial application of plant cell cultures is often hindered by the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and decline dramatically over subcultures. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesig, intracellular carbon, nitrogen and phosphate during continuous 10 subculture cycles were investigated. The subculture cycle was fluctuated for 12h in a 7 day cycle (6.5, 7 and 7.5 d), and the inoculum size was fluctuated by 20% on basis of 2.00 g (1.60, 2.00 and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7d-subculture cycle and 1.60 g-inoculum size was the best one to hold the relatively stable anthocyanin production. The anthocyanin yield presented a negative correlation with intracellular sucrose content or intracellular total phosphate content.

  13. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  14. Methyl Jasmonate Represses Growth and Affects Cell Cycle Progression in Cultured Taxus Cells

    OpenAIRE

    Patil, Rohan A.; Lenka, Sangram K.; Normanly, Jennifer; Walker, Elsbeth L.; Roberts, Susan C.

    2014-01-01

    Methyl jasmonate (MeJA) elicitation is an effective strategy to induce and enhance synthesis of the anticancer agent paclitaxel (Taxol®) in Taxus cell suspension cultures; however, concurrent decreases in growth are often observed, which is problematic for large scale bioprocessing. Here, increased accumulation of paclitaxel in Taxus cuspidata suspension cultures with MeJA elicitation was accompanied by a concomitant decrease in cell growth, evident within the first three days post-elicitatio...

  15. Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures.

    Science.gov (United States)

    Fox, Emily P; Cowley, Elise S; Nobile, Clarissa J; Hartooni, Nairi; Newman, Dianne K; Johnson, Alexander D

    2014-10-20

    The human microbiome contains diverse microorganisms, which share and compete for the same environmental niches. A major microbial growth form in the human body is the biofilm state, where tightly packed bacterial, archaeal, and fungal cells must cooperate and/or compete for resources in order to survive. We examined mixed biofilms composed of the major fungal species of the gut microbiome, Candida albicans, and each of five prevalent bacterial gastrointestinal inhabitants: Bacteroides fragilis, Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. We observed that biofilms formed by C. albicans provide a hypoxic microenvironment that supports the growth of two anaerobic bacteria, even when cultured in ambient oxic conditions that are normally toxic to the bacteria. We also found that coculture with bacteria in biofilms induces massive gene expression changes in C. albicans, including upregulation of WOR1, which encodes a transcription regulator that controls a phenotypic switch in C. albicans, from the "white" cell type to the "opaque" cell type. Finally, we observed that in suspension cultures, C. perfringens induces aggregation of C. albicans into "mini-biofilms," which allow C. perfringens cells to survive in a normally toxic environment. This work indicates that bacteria and C. albicans interactions modulate the local chemistry of their environment in multiple ways to create niches favorable to their growth and survival.

  16. The use of morphogenic suspension cultures for the development of a protoplast regeneration system in lily

    NARCIS (Netherlands)

    Famelaer, L.; Bordas, M.; Baliu', E.; Ennik, E.; Meijer, H.; Tuyl, van J.M.; Creemers-Molenaar, J.

    1997-01-01

    The present study reports data on the development of a protoplast regeneration procedure in lily. Established morphogenic suspension cultures were obtained from callus cultures induced on mature embryos from crosses between cultivars of L. longiflorum. The effect on the frequency of protoplast divis

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

    Directory of Open Access Journals (Sweden)

    Jadwiga H. Supniewska

    2015-05-01

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

  18. A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

    Science.gov (United States)

    Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

    2015-09-01

    A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed. PMID:26548080

  19. 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...

  20. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

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

  1. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

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

  2. 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

  3. Improved Production of Paclitaxel from Suspension Culture of Taxus chinensis var.mairei by in situ Extraction with Organic Solvents

    Institute of Scientific and Technical Information of China (English)

    未作君; 元英进; 吴兆亮; 吴金川

    2003-01-01

    The production of paclitaxel from suspension culture of Taxus chinensis var,mairei was improved by in situ extraction with organic solvents to avoid feedback repression and product degradation.Oleic acid and dibutyl phthalate were proved to be suitable solvents .The optimal volumetric percentage of organic solvents in the culture medium was found to be around 8%,and the favorable time for their introduction was at the exponential phase of cell growth,Paclitaxel production with the in situ extraction was ca 3-fold of that without extraction.

  4. A genotype of modified vaccinia Ankara (MVA) that facilitates replication in suspension cultures in chemically defined medium.

    Science.gov (United States)

    Jordan, Ingo; Horn, Deborah; John, Katrin; Sandig, Volker

    2013-01-21

    While vectored vaccines, based on hyperattenuated viruses, may lead to new treatment options against infectious diseases and certain cancers, they are also complex products and sometimes difficult to provide in sufficient amount and purity. To facilitate vaccine programs utilizing host-restricted poxviruses, we established avian suspension cell lines (CR and CR.pIX) and developed a robust, chemically defined, culturing process for production of this class of vectors. For one prominent member, modified vaccinia Ankara (MVA), we now describe a new strain that appears to replicate to greater yields of infectious units, especially in the cell-free supernatant of cultures in chemically defined media. The new strain was obtained by repeated passaging in CR suspension cultures and, consistent with reports on the exceptional genetic stability of MVA, sequencing of 135 kb of the viral genomic DNA revealed that only three structural proteins (A3L, A9L and A34R) each carry a single amino acid exchange (H639Y, K75E and D86Y, respectively). Host restriction in a plaque-purified isolate of the new genotype appears to be maintained in cell culture. Processing towards an injectable vaccine preparation may be simplified with this strain as a complete lysate, containing the main burden of host cell contaminants, may not be required anymore to obtain adequate yields.

  5. 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...

  6. Optimizing stem cell culture.

    OpenAIRE

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-01-01

    International audience Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such a...

  7. Digital Microfluidic Cell Culture.

    Science.gov (United States)

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  8. Automated single cell isolation from suspension with computer vision

    OpenAIRE

    Rita Ungai-Salánki; Tamás Gerecsei; Péter Fürjes; Norbert Orgovan; Noémi Sándor; Eszter Holczer; Robert Horvath; Bálint Szabó

    2016-01-01

    Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any...

  9. Generation of a MDCK Cell Line to Suspension Culture by Stably Experessing Human Siat7e Gene%稳定表达人Siat7e基因的MDCK细胞系的建立和全悬浮细胞的驯化

    Institute of Scientific and Technical Information of China (English)

    张妍; 张文俊; 李群辉; 刘晓文; 刘文博; 刘秀梵

    2011-01-01

    目的:现有的禽流感疫苗生产的方法已经不能适应工业化大生产的需求,有必要开发全悬浮培养的细胞系来满足大生产的需求.方法:我们通过转染稳定表达Siat7e基因的真核表达载体对MDCK细胞进行改造,并经过后期的驯化,筛选适应于全悬浮培养的MDCK细胞.结果:成功筛选到能稳定表达Siat7e基因并能适应全悬浮生长的细胞系.结论:该细胞系具有潜在的应用价值,为MDCK细胞的培养以及工业化大生产疫苗提供参考.%Object; Current influenza vaccine manufacturing have been unable to meet the demand of Industrialized production. Method; Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human Siat7e gene (ST6GalNac V). Result: Successfully conversioned a MDCK cell line stably experessing human Siat7e and they were capable of growing in suspension cultures. Conclusion: These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale vaccine production.

  10. Brief Azacytidine Step Allows The Conversion of Suspension Human Fibroblasts into Neural Progenitor-Like Cells

    Directory of Open Access Journals (Sweden)

    Fahimeh Mirakhori

    2015-04-01

    Full Text Available In recent years transdifferentiation technology has enabled direct conversion of human fibroblasts to become a valuable, abundant and accessible cell source for patient-specific induced cell generation in biomedical research. The majority of transdifferentiation approaches rely upon viral gene delivery which due to random integration with the host genome can cause genome instability and tumorigenesis upon transplantation. Here, we provide a simple way to induce neural progenitor-like cells from human fibroblasts without genetic manipulation by changing physicochemical culture properties from monolayer culture into a suspension in the presence of a chemical DNA methyltransferase inhibitor agent, Azacytidine. We have demonstrated the expression of neural progenitor-like markers, morphology and the ability to spontaneously differentiate into neural-like cells. This approach is simple, inexpensive, lacks genetic manipulation and could be a foundation for future chemical neural transdifferentiation and a safe induction of neural progenitor cells from human fibroblasts for clinical applications.

  11. Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

    Institute of Scientific and Technical Information of China (English)

    Xing Yang; Zhaoying Zhou; Mingfei Xiao; Ying Wu; Shangfeng Liu

    2006-01-01

    A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed. The substrate and the electrodes of the chip were made of glass and gold, respectively. The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline, culture medium, living cell suspension etc.) by scanning from 10Hz to 45kHz. A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension. An actual circuit was also built and tested to verify the 6-element circuit model proposed. The micro-EIS chip has several advantages including the use of small sample volumes, high resolution and ease of operation. It shows good application prospects in the areas of cellular electrophysiology, drug screening and bio-sensors etc.

  12. Comparison of Different Culture Mode for Long-term Expansion of Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 Introduction The mammalian central nervous system(CNS) is incredibly complex and possesses only a limited ability to recover from damage~([1]). Fortunately, the discovery of self-renewing stem cell populations within the fetal and adult CNS has opened promising lines of inquiry. Neural stem cells (NSCs) can be cultured in two modes in vitro, suspension and monolayer. Reynolds~([2]) and other groups culture NSCs as neurospheres in suspension. Alternatively Gage cultured NSCs in monolayer. There is little s...

  13. Dialysis buffer with different ionic strength affects the antigenicity of cultured nervous necrosis virus (NNV) suspensions.

    Science.gov (United States)

    Gye, Hyun Jung; Nishizawa, Toyohiko

    2016-09-01

    Nervous necrosis virus (NNV) belongs to the genus Betanodavirus (Nodaviridae). It is highly pathogenic to various marine fishes. Here, we investigated the antigenicity changes of cultured NNV suspensions during 14days of dialyses using a dialysis tube at 1.4×10(4) molecular weight cut off (MWCO) in three different buffers (Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), and deionized water (DIW)). Total NNV antigen titers of cultured NNV suspension varied depending on different dialysis buffers. For example, total NNV antigen titer during D-PBS dialysis was increased once but then decreased. During Tris-HCl dialysis, it was relatively stable. During dialysis in DIW, total NNV antigen titer was increased gradually. These antigenicity changes in NNV suspension might be due to changes in the aggregation state of NNV particles and/or coat proteins (CPs). ELISA values of NNV suspension changed due to changing aggregates state of NNV antigens. NNV particles in suspension were aggregated at a certain level. These aggregates were progressive after D-PBS dialysis, but regressive after Tris-HCl dialysis. The purified NNV particles self-aggregated after dialysis in D-PBS or in Tris-HCl containing 600mM NaCl, but not after dialysis in Tris-HCl or DIW. Quantitative analysis is merited to determine NNV antigens in the highly purified NNV particles suspended in buffer at low salt condition. PMID:27381060

  14. 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.

  15. 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.

  16. Growth inhibition by exogenous proline and its metabolism in saltgrass (Distichlis spicata) suspension cultures.

    Science.gov (United States)

    Rodriguez, M M; Heyser, J W

    1988-08-01

    The growth of Distichlis spicata suspension cultures in LS medium without NaCl was inhibited 54% by 2 mM proline. In medium containing 260 mM NaCl, 10 mM proline inhibited growth by only 22%. The uptake and metabolism of 10 mM L-[1-(13)C] proline was followed by (13)C NMR and ninhydrin analyses of suspensions cultured in the presence of 0 or 260 mM NaCl. Uptake of 85 to 92% of the exogenous proline occurred within 72 h in all media. In 10 mM proline and no NaCl, cellular proline reached a maximm of 51.5 μmoles/g FW compared to 1.9 μmoles/g FW in suspensions not grown on proline. In medium containing 260 mM NaCl and proline, cellular proline reached 59-65 μmoles/g FW compared to 30-40 μmoles/g FW in controls grown without proline. The (13)C-label in the proline-C1 was either retained in proline or disappeared, presumably released as carbon dioxide, by catabolism through the TCA cycle. Since no metabolite of (13)C-proline was detected by NMR, proline was considered to be the molecule which inhibited the suspension culture growth.

  17. Effects of elicitors on saffron pigment production in cell suspension cultures of Crocus sativus L.%诱导子对藏红花悬浮培养细胞生产藏红花色素的影响

    Institute of Scientific and Technical Information of China (English)

    孙镇; 袁丽红; 吴频梅

    2013-01-01

    The effects of chitosan,chitosan oligosaccharides (COS),methyl jasmonate (MJ),salicylic acid (SA),and copper ions(Cu2+) on cell growth and saffron pigment production in cell suspension cultures of C.sativus were investigated.The results showed that COS(1-500 mg/L),chitosan(≤ 10 mg/L),M J(≤ 10μmol/L),SA(≤10 μmol/L),and Cu2+ (≤1 μmol/L) had no significant effects on saffron cell growth,whereas chitosan(≥100 mg/L),MJ(≥100 μmol/L),SA(≥ 100 μmol/L),and Cu2+ (≥ 10 μmol/L) at high concentrations obviously inhibited the cell growth.The effects of elicitors were significant on saffron pigment biosynthesis,and had relations with the concentrations of elicitors and the timing of elicitation.MJ was the best elicitor,the content of saffron pigment reached 28.57 mg/g DW by adding 100 μmol/L MJ on day 0,and it was higher by 177.9% than the control.Cu2+ was the second,the content of saffron pigment reached 19.82 mg/g DW by adding 500 μmol/L Cu2 + on day 4,and it was higher by 108.2% than the control.Chitosan and COS were the third,the contents of saffron pigment reached 18.33 mg/gDW and 17.39 mg/gDW by adding 100 mg/L chitosan and oligochitosan on day 14,and it was higher by 69.1% and 69.0% than the control,respectively.The last one is SA,the content of saffron pigment reached 14.65mg/gDW by adding 10 μmol/L SA on day 14 and it was higher by 45.4% than the control.%考察壳聚糖(chitosan)、壳寡糖(chitosan oligosaccharides,COS)、茉莉酸甲酯(methyl jasmonate,MJ)、水杨酸(salicylic acid,SA)和Cu2+等诱导子对藏红花悬浮培养细胞生长和藏红花色素合成的影响.结果表明:在实验考察浓度范围内,壳寡糖(1 ~ 500 mg/L)和较低浓度壳聚糖(≤10 mg/L)、MJ(≤10 μmol/L)、SA(≤10 μmol/L)和Cu2+(≤1 μmol/L)对细胞生长无显著影响;较高浓度壳聚糖(≥100 mg/L)、MJ(≥100 μmol/L)、SA(≥100 μmol/L)和Cu2+(≥10 μmol/L)显著抑制细胞生长.5种诱导子对藏红花色素合成的

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

    Directory of Open Access Journals (Sweden)

    Zofia Chirek

    2014-02-01

    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.

  19. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    Science.gov (United States)

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. PMID:25879277

  20. [CO-CULTURE OF BOAR SPERMATOGONIAL CELLS WITH SERTOLI CELLS].

    Science.gov (United States)

    Savchenkova, I P; Vasil'eva, S A

    2016-01-01

    In the present study, we developed in vitro culture conditions using co-culture of boar spermatogonial cells with Sertoli cells. Testes from 60-day-old crossbred boar were used. A spermatogonia-enriched culture was achieved by enzymatic digestion method and purification by density gradient centrifugation using a discontinuous Percoll gradient and differentiated adherence technique. Lipid drops were detected in isolated Sertoli cells by Oil Red O staining. We have found that the cultivation of boar spermatogonia in the presence of Sertoli cells (up to 35 days) leads to their differentiation as well as in vivo in testis. Association of cells in groups, formation of chains and suspension clusters of the spermatogenic cells were observed on the 10th day. Spermatogonial cellular colonies were noted at the same time. These cellular colonies were analyzed for the expression of genes: Nanog and Plzf in RT PCR. The expression of the Nanog gene in the experimental cellular clones obtained by short-term culture of spermatogonial cells in the presence of Sertoli cells was 200 times higher than the expression of this gene in the freshly isolated spermatogonial cells expression was found in freshly isolated germ cells and in cellular clones derived in vitro. We have found that, in the case of longer cultivation of these cells on Sertoli cells, in vitro process of differentiation of germ cells and formation of single mobile boar spermatozoa occurs at 30-33 days. Cellular population is heterogeneous at this stage. Spermatogenic differentiation in vitro without Sertoli cells stays on the 7th day of cultivation. The results show that co-culture of boar spermatogonia-enriched cells with Sertoli cells can induce their differentiation into spermatozoa in vitro and facilitate obtaining of porcine germ cell culture. PMID:27228660

  1. [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.

  2. Out-of-School Suspensions of Black Youths: Culture, Ability, Disability, Gender, and Perspective.

    Science.gov (United States)

    Haight, Wendy; Kayama, Misa; Gibson, Priscilla Ann

    2016-07-01

    Racial disproportionality in out-of-school suspensions is a persistent social justice issue in public schools. This article examines out-of-school suspensions of four black youths from the perspectives of the youths, their caregivers, and educators. The case involving David, a 14-year-old African American with a learning disability, illustrates the challenges of students experiencing the intersection of disability and race. The case involving George, a 14-year-old Liberian immigrant, illustrates how parents and teachers may form alliances around shared goals and values despite profound cultural differences in understanding of youths' misbehavior. The case involving Nina, a 12-year-old African American, illustrates how educators' failure to consider the context of her misbehaviors as responses to sexual harassment, along with their subsequent harsh punishment and failure to protect her, led to her disengagement from school. The case involving Craig, a 16-year-old African American, provides a glimpse into how the use of criminal justice language to refer to youths' misbehaviors can support the development of a criminalized self- and social identity. These cases illustrate the diversity of black students--including ability, disability, culture, and gender--and how events surrounding suspensions are interpreted by students, caregivers, and educators. Understanding such diversity will undergird implementation of effective alternatives to suspensions. PMID:27501641

  3. Out-of-School Suspensions of Black Youths: Culture, Ability, Disability, Gender, and Perspective.

    Science.gov (United States)

    Haight, Wendy; Kayama, Misa; Gibson, Priscilla Ann

    2016-07-01

    Racial disproportionality in out-of-school suspensions is a persistent social justice issue in public schools. This article examines out-of-school suspensions of four black youths from the perspectives of the youths, their caregivers, and educators. The case involving David, a 14-year-old African American with a learning disability, illustrates the challenges of students experiencing the intersection of disability and race. The case involving George, a 14-year-old Liberian immigrant, illustrates how parents and teachers may form alliances around shared goals and values despite profound cultural differences in understanding of youths' misbehavior. The case involving Nina, a 12-year-old African American, illustrates how educators' failure to consider the context of her misbehaviors as responses to sexual harassment, along with their subsequent harsh punishment and failure to protect her, led to her disengagement from school. The case involving Craig, a 16-year-old African American, provides a glimpse into how the use of criminal justice language to refer to youths' misbehaviors can support the development of a criminalized self- and social identity. These cases illustrate the diversity of black students--including ability, disability, culture, and gender--and how events surrounding suspensions are interpreted by students, caregivers, and educators. Understanding such diversity will undergird implementation of effective alternatives to suspensions.

  4. 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....

  5. Acaricidal activities of whole cell suspension, cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

    Institute of Scientific and Technical Information of China (English)

    Prapassom BUSSAMAN; Chirayu SA-UTH; Paweena RATTANAS ENA; Angsumarn CHANDRAPATYA

    2012-01-01

    Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp.,a mushroom mite endemic in Thailand.To develop an effective formulation of Xenorhabdus stokiae,treatments using different parts of X.stokiae isolate PB09 culture,including whole cell suspension,cell-free supernatant,and crude cell extract,were performed.The results show that different parts ofX.stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity.Application with cell-free supernatant of X.stokiae culture resulted in both the highest mite mortality rate [(89.00+3.60)%] and the lowest mite fecundity [(41.33+23.69) eggs/gravid female].Whole cell suspension of X.stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant,suggesting that X.stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media.Crude cell extract of X.stokiae was not effective against mites.Cell-free supernatant of X.stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

  6. 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.

  7. 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.

  8. Suspended Cell Culture ANalysis (SCAN) Tool to Enhance ISS On-Orbit Capabilities Project

    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,...

  9. Enhancement of heat transfer in red cell suspensions in vitro experiments.

    Science.gov (United States)

    Carr, R T; Tiruvaloor, N R

    1989-05-01

    New data on laminar heat convection with red cell suspensions have been gathered for both heating and cooling. When compared to data for the suspending medium alone, it is apparent that the red cells enhance laminar heat transfer when Pe greater than 4. This is probably due to particle movements. These new data disagree with earlier studies which indicated no enhancement of heat transfer for blood cell suspensions. The data do agree with previous correlations for enhanced thermal transport in sheared suspensions.

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

    Science.gov (United States)

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

    2006-10-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). 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 of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.

  11. Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

    OpenAIRE

    Hu, Suwen; Deng, Lei; Wang, Huamao; Zhuang, Yingping; Chu, Ju; Zhang, Siliang; Li, Zhonghai; Guo, Meijin

    2011-01-01

    The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 105 cells/mL. Then, the basic metabolic ...

  12. Extracted hair follicle outer root sheath cell suspension for pigment cell restoration in vitiligo

    Directory of Open Access Journals (Sweden)

    Anil Kumar

    2013-01-01

    Full Text Available Vitiligo surgery has come up a long way from punch skin grafts to epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension (EHF-ORS-CS transplantation. The progressive development from one technique to the other is always in a quest for the best. In the latest development- EHF-ORS-CS, which is an enriched source of follicular inactive melanocyte (melanocyte stem cells, seems to be a good addition to the prevailing cell-based therapies for vitiligo; however, need to be explored further in larger, and preferably randomized blinded studies. This review discusses the principle, technical details, and stem cell composition of hair follicular outer root sheath cell suspension.

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

    International Nuclear Information System (INIS)

    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)

  14. Cell suspension as a tool to study the biosynthesis of pilocarpine in Jaborandi.

    Science.gov (United States)

    Abreu, I N; Andreazza, N L; Sawaya, A C H F; Eberlin, M N; Mazzafera, P

    2007-11-01

    Jaborandi (Pilocarpus microphyllus) is a species that naturally occurs in the North and Northeast of Brazil, whose leaves produce pilocarpine (an imidazole alkaloid that has been used to treat glaucoma and xerostomy), the biosynthesis of which is still uncertain. The aim of this work was to establish cell lineages and select them according to an alkaloid profile similar to the one from Jaborandi leaves. The induction of callus was done in different culture media and growth regulators. Calluses from primary cultures or those subcultured several times were used as explants for the obtainment of six cell lineages. Alkaloids content analyses and growth curves showed that lines obtained from primary cultures produced more alkaloids and a better development. Cell lines from 12 subcultures presented a decrease in pilocarpine and pilosine production. After 24 subcultures, the production of alkaloids remained constant. ESI-MS analysis showed that cell culture extracts have the same alkaloid composition as extracts made from leaves. The results indicate that cell suspensions can be used as a model to study the biosynthesis of the imidazole alkaloid in P. microphyllus. PMID:17682964

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

    OpenAIRE

    Z. Tofighi; Ghazi saeidi; A. Hadjiakhoondi; Yassa, N.

    2016-01-01

    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...

  16. Metal supported tubular solid oxide fuel cells fabricated by suspension plasma spray and suspension high velocity oxy-fuel spray

    Science.gov (United States)

    Yoo, Yeong; Wang, Youliang; Deng, Xiaohua; Singh, Devinder; Legoux, Jean-Gabriel

    2012-10-01

    Low temperature (LT) metal supported solid oxide fuel cells (SOFCs) have many advantages in comparison to conventional electrode or electrolyte supported type SOFCs. NRC has demonstrated high performance LT metal supported planar SOFCs fabricated by either wet colloidal spray/sintering or suspension thermal spray. The combination of tubular configuration and metal supported SOFCs may produce more unique and very attractive advantages such as easy and inexpensive sealing method and materials, high specific and volumetric power density, cost-effective fabrication, enhanced robustness, rapid start up, red-ox cycle tolerance and potential use for a pressurized integrated system. In this paper, thin film solid electrolyte of Sm0.2Ce0.8O1.90 (SDC) and NiO-SDC composite anode on sintered porous tubular metal supports were deposited by suspension HVOF spray and suspension plasma spray, respectively on sintered porous tubular metal support. La0.6Sr0.4Co0.2Fe0.8O3-δ (LSCF) cathode on the SDC electrolyte was formed by wet colloidal spray and subsequent sintering process as the final fabrication step. The detailed investigation of suspension and process-related parameters for suspension thermal spray was performed in order to produce thin and crack-free SDC thin film coatings. The electrochemical performance of single cells was demonstrated.

  17. Polyphosphoinositides are present in plant tissue culture cells

    International Nuclear Information System (INIS)

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

  18. 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. PMID:27108314

  19. Establishment of long-term monocyte suspension cultures from normal human peripheral blood

    OpenAIRE

    1982-01-01

    The long-term suspension growth of normal, immature myeloid cells from fresh human cord blood was recently reported and required cells separated on supplemented discontinuous Percoll gradients, growth in media containing hydrocortisone and vitamins D3, and gentle, continuous agitation (13). When normal adult bone marrow (six donors) or blood from Epstein-Barr virus (EBV)-seropositive donors (nine donors) was used as a source of fresh human leukocytes, only short-term proliferation of myeloid ...

  20. 超声、DMSO和CMC-Na处理对喜树细胞悬浮培养的影响%Influences of ultrasonic wave,DMSO and CMC-Na on cell suspension cul-tures of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    康大力; 夏黎; 张洪利

    2014-01-01

    目的:研究超声及添加细胞分泌促进剂、培养液增稠剂对喜树细胞悬浮培养的影响。方法采用不同超声时间、不同浓度的 DMSO 和CMC-Na作用于悬浮生长的喜树细胞,观察喜树细胞的生长情况。结果10s 超声波作用及0.5% DMSO 可显著增加细胞生物量,添加1%CMC-Na培养的喜树细胞几乎呈单细胞状态。结论施加适当的超声、DMSO和CMC-Na处理可显著影响悬浮培养喜树细胞生物量和生长状态。%OBJECTIVE To study the infulences of ultrasonic wave ,permeabilization and viscous additive-sup-plemented medium on cell cultures of Camptotheca acuminata.METHODS The cell cultures were treated with dif-ferent durations of ultrasonic wave and different concentrations of DMSO and CMC-Na and the growth rates of cells were investigated.RESULTS Ten seconds of ultrasonic wave and 0.5%DMSO could significantly improve the cell biomass and the cells grew seperately when the medium was added with 1%CMC-Na.CONCLUSION Appropriate ultrasonic wave ,DMSO and CMC-Na can significantly affect the biomass and cell status of Camptotheca acuminata.

  1. Preparation of Single Cell Suspensions from Mouse Aorta

    Science.gov (United States)

    Hu, Desheng; Yin, Changjun; Mohanta, Sarajo K.; Weber, Christian; Habenicht, Andreas J. R.

    2016-01-01

    Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner et al., 2009; Hu et al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa et al., 2016; Galkina et al., 2006; Lotzer et al., 2010; Mohanta et al., 2016; Mohanta et al., 2014; Moos et al., 2005; Srikakulapu et al., 2016; Zhao et al., 2004). There are conflicting reports on the number and subtypes of immune cells in the aorta depending on the age of the animals, the protocol that is used to obtain single cell suspensions, and the dietary conditions of the mice (Campbell et al., 2012; Clement et al., 2015; Galkina et al., 2006; Kyaw et al., 2012). The number of immune cells in the aorta differs as much as tenfold using different protocols (Butcher et al., 2012; Galkina et al., 2006; Gjurich et al., 2015; Grabner et al., 2009; Hu et al., 2015). These discrepant results call for a protocol that robustly documents bona fide aorta cells rather than those in the surrounding tissues or blood. Critical methodological hurdles include the removal of adjacent adipose tissue and small paraaortic lymph nodes lining the entire aortic tree that are not visible by the naked eye. A dissection microscope is therefore recommended. Moreover protocols of aorta preparations should ascertain that lymphocyte aggregates referred to as fat associated lymphoid clusters (FALCs) (Benezech et al., 2015; Elewa et al., 2015) that are often present at the border between the adipose tissue and the adventitia are removed before enzyme digestion. We propose - besides other approaches (Hu et al., 2015; Mohanta et al., 2014) - a combination of immunohistochemical staining and

  2. Cell culture's spider silk road.

    Science.gov (United States)

    Perkel, Jeffrey

    2014-06-01

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

  3. 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.

  4. 几种物理因素对黄芩细胞悬浮系生长与黄芩苷含量变化的影响%Effects of some physiocal factors on growth of cell suspension culture from Scutellaria baicalins and changes of baicalin content

    Institute of Scientific and Technical Information of China (English)

    顾长海; 张东向; 刘丽杰; 赵静; 卢蓉蓉; 王洪杰; 佟鑫鑫

    2014-01-01

    For establish stable cell suspension skullcap,cell culture technique were used to study the three kinds of physical factors such as inoculation,light and pH on change of growth of Scutellaria suspension cell lines and the baicalin content. The results showed that the turbidity of skullcap cell suspension presented"S"shaped curve with incubation time. Using MS basic medium , add 0.2 mg · L-1 2,4-D, 2.0 mg · L-1 6-BA and 3 % sucrose , 1.0g·L-1 of inoculum,pH5.8,and incubated at 25℃dark conditions most conducive to the accumulation of baicalin, and baicalin up to 7.425μg·g-1FW.%为建立稳定的黄芩细胞悬浮系,采用悬浮细胞培养技术,研究了接种量、光照与否和pH值等3种物理因素对黄芩细胞悬浮系生长与黄芩苷含量变化的影响。结果表明,黄芩细胞悬浮系浊度随培养时间呈现“S”形变化曲线;采用MS基本培养基,添加0.2 mg·L-12,4-D、2.0 mg·L-16-BA 和3%蔗糖,选取1.0 g·L-1的接种量、pH 5.8,并在25℃条件下进行暗培养,最有利于黄芩苷的积累,黄芩苷含量最高可达7.425μg·g-1FW。

  5. Development, characterization, and optimization of a new suspension chicken-induced pluripotent stem cell line for the production of Newcastle disease vaccine

    Science.gov (United States)

    Traditionally, substrates for production of vaccines have been embryonated eggs or adherent cell culture. The daunting challenge of scaling up these technologies in the face of an outbreak has been a limitation for industrial applicability. Suspension cell lines are better suited in many ways to e...

  6. Withaferin A from cell cultures of Withania somnifera

    Directory of Open Access Journals (Sweden)

    Ciddi Veeresham

    2006-01-01

    Full Text Available Suspension cultures of Withania somnifera cells were established and shown to produce withaferin A. The identification of withaferin A was done by TLC, UV absorption, HPLC and electron spray mass spectroscopy. These cultures could be strongly elicited by exposure to salacin. Addition of salacin at the concentration of 750 µM to the cultures in production medium enhanced production levels of withaferin A to 25±2.9 mg/l compared to 0.47±0.03 mg/l in unelicited controls. This report is the first to demonstrate withaferin A production in plant suspension cultures and provides prerequisites for commercial scale, controlled production of withaferin A.

  7. Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen.

    Science.gov (United States)

    Hamerski, D; Matern, U

    1988-01-15

    Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome-P450-dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O-prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.

  8. 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

  9. An Application of Microcapsules Having Enzyme-degradable Gel Membrane to Cell Culture

    Science.gov (United States)

    Dobashi, Toshiaki; Koike, Michiru; Kobayashi, Kentaro; Maki, Yasuyuki; Yamamoto, Takao; Tanaka, Susumu

    Newly developed microcapsules having gelatin wall membrane was applied as a scaffold for suspension cell culture. The optimum preparation condition was determined, and the stability of the cultured human fibroblast cells using the microcapsules was examined at both protein and gene levels.

  10. Effect of endophytic fungal elicitors on growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea%内生真菌诱导子对茅苍术细胞生长及苍术素积累的影响

    Institute of Scientific and Technical Information of China (English)

    陶金华; 濮雪莲; 江曙

    2011-01-01

    目的:研究内生真菌诱导子对茅苍术细胞生长以及苍术素积累的影响.方法:采用细胞悬浮培养方法研究内生真菌诱导子不同浓度、不同加入时间对茅苍术细胞生长、苍术素合成以及相关酶活性的影响.结果:低浓度诱导子对细胞生长没有明显的影响,但高浓度诱导子显著抑制细胞生长,当诱导子质量浓度达到100 mg·L-1时,细胞生长抑制率达到46.7%;此外,从茅苍术内生真菌中筛选获得6株具有较强诱导作用的内生真菌,其中Rhizoctonia SP1诱导子的作用较强,20~60 mg·L-1 Rhizoctonia SP1诱导子显著促进苍术素的生物合成.在细胞培养至12 d时,添加40 mg·L-1 Rhizoctonia SP1诱导子,苍术素的含量在第21天达到最大值28.06μg·L-1,比对照提高了48.3%;且细胞多酚氧化酶(PPO)、过氧化物酶(POD)、过氧化氢酶(CAT)的活性出现了显著提高.结论:内生真菌诱导子能有效提高茅苍术细胞悬浮培养体系中苍术素的产量.%Objective: To investigate the effects of endophytic fungal elicitors on the growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea. Method: The endophytic fungal elicitors were added to the medium with different concentrations and culture period. Their effects on biomass, atractylodin content and relevant enzyme activities in suspension cultured cells were studied. Result: The cell growth was not affected by elicitors at low concentration and obviously inhibited at high concentration.Inhibition rate reached 46.7% by 100 mg· L-1 elicitor. In addition, six strains from A. lancea, among which Rhizoctonia SP1 activity was higher, had distinctly promoted the accumulation of atractylodin. Atractylodin biosynthesis was notably promoted by 20-60 mg·L-1 Rhizoctonia SP1 elicitor. When 40 mg · L-1 Rhizoctonia SP1 elicitor was added in the medium at the 12 day, the maximum content of atractylodin was 28.06 μg · L-1 at the 21 day with 48.3

  11. The establishment of suspension and meristem cultures for the development of a protoplast regeneration and fusion system in Lily

    NARCIS (Netherlands)

    Famelaer, I.; Ennik, E.; Tuyl, van J.M.; Meijer, H.; Creemers-Molenaar, J.

    1996-01-01

    The present results indicate that established morphogenic suspension cultures can be obtained from crosses between cultivars of L. longiflorum and from a cross between Asiatic hybrid 'Orlito' x 'Connecticut King'. Meristem cultures were obtained from L. longiflorum 'Gelria' and Oriental hybrid 'Star

  12. 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.

  13. Effects of lovastatin, clomazone and methyl jasmonate treatment on the accumulation of purpurin and mollugin in cell suspension cultures of Rubia cordifolia%洛伐他丁、氯胺和茉莉酸甲酯处理对茜草悬浮培养细胞中紫色素和大叶茜草素积累的作用

    Institute of Scientific and Technical Information of China (English)

    樊星; 胡高升; 李娜; 韩志福; 贾景明

    2013-01-01

    目的:使用茜草细胞悬浮培养体系研究茜草中萘醌类成分生合成途径原料IPP的来源,以及茉莉酸甲酯、氯胺和洛伐他丁对蒽醌和萘醌的积累情况.方法:建立茜草细胞悬浮培养体系,分别在培养基中添加茉莉酸甲酯、氯胺和洛伐他丁.每3天取样一次.使用高效液相色谱法测定紫色素和大叶茜草素含量并计算出产量.结果:洛伐他丁处理组中紫色素和大叶茜草素产量均有显著增加.氯胺处理组中紫色素和大叶茜草素产量明显下降.茉莉酸甲酯处理组中紫色素产量增加,同时大叶茜草素产量降低.结论:在茜草细胞中萘醌类成分的IPP可能是通过MEP途径合成的;要解释茉莉酸甲酯对蒽醌和萘醌积累的不同作用,基因表达水平方面的研究尤其是对1,4-二羟基-2-萘甲酸异戊烯基化的基因表达研究应该受到更多关注.%AIM:To determine the IPP origin of the naphthoquinones (NQs) in Rubia cordifolia,and to evaluate the effects of methyl jasmonate (MeJA) treatment,MEP,and MVA pathway inhibitor treatment on the accumulation of anthraquinones (AQs) and NQs in cell suspension cultures of R.cordifolia.METHODS:Cell suspension cultures of R.cordifolia were established.Specific inhibitors (lovastatin and clomazone) and MeJA were supplied to the media,respectively.Treated cells were sampled every three days.Content determination of purpurin (AQs) and mollugin (NQs) were carried out using RP-HPLC.The yield of the two compounds was compared with the DMSO-supplied group and the possible mechanism was discussed.RESULTS:Lovastatin treatment increased the yield of purpurin and mollugin significantly.Clomazone treatment resulted in a remarkable decrease of both compounds.In the MeJA-treated cells,the purpurin yield increased,meanwhile,the mollugin yield decreased compared with control.CONCLUSION:the IPP origin of mollugin in R.cordifolia cell suspension cultures was likely from the MEP pathway.To explain

  14. The durative use of suspension cells and callus for volatile oil by comparative with seeds and fruits in Capparis spinosa L.

    Directory of Open Access Journals (Sweden)

    Yongtai Yin

    Full Text Available Capparis spinosa is one of the most important eremophytes among the medicinal plants, and continued destruction of these plants poses a major threat to species survival. The development of methods to extract compounds, especially those of medicinal value, without harvesting the whole plant is an issue of considerable socioeconomic importance. On the basis of an established system for culture of suspension cells and callus in vitro, Gas Chromatograph-Mass Spectrometer (GC-MS was used for the volatile oil composition analyzing in seed, fruit, suspension cells and callus. Fatty acids were the major component, and the highest content of alkanes was detected in seed, with <1.0% in suspension cells and callus. Esters, olefins and heterocyclic compounds were significantly higher in fruit than in the other materials. The content of acid esters in the suspension cells and callus was significantly higher than in seed and fruit. This indicated that the suspension cells and callus could be helpful for increasing the value of volatile oil and replacing seeds and fruit partially as a source of some compounds of the volatile oil and may also produce some new medical compounds. The above results give valuable information for sustainable use of C. spinosa and provide a foundation for use of the C. spinosa suspension cells and callus as an ongoing medical resource.

  15. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

    Directory of Open Access Journals (Sweden)

    Liangyun Zhou

    2016-09-01

    Full Text Available Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE treatment. An ultra-performance liquid chromatography (UPLC method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS LC−MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2′-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 μg·g−1 at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-d-glucopyranoside. The content of 2′-Hydroxyaucuparin reached its highest (422.75 μg·g−1 at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

  16. Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani.

    Science.gov (United States)

    Venugopalan, Aarthi; Srivastava, Smita

    2015-01-01

    Ethanolic extract of a non-camptothecin producing plant, Catharanthus roseus when added in the suspension culture of the endophyte Fusarium solani known to produce camptothecin, resulted in enhanced production of camptothecin by 10.6-fold in comparison to that in control (2.8 μg/L). Interestingly, addition of pure ethanol (up to 5% v/v) in the suspension culture of F. solani resulted in maximum enhancement in camptothecin production (up to 15.5-fold) from that obtained in control. In the presence of ethanol, a reduced glucose uptake (by ∼ 40%) and simultaneous ethanol consumption (up to 9.43 g/L) was observed during the cultivation period (14 days). Also, the total NAD level and the protein content in the biomass increased by 3.7- and 1.9-fold, respectively, in comparison to that in control. The study indicates a dual role of ethanol, presumably as an elicitor and also as a carbon/energy source, leading to enhanced biomass and camptothecin production. PMID:25603728

  17. Extracellular Hydrolysis of Starch in Sugarcane Cell Suspensions 12

    Science.gov (United States)

    Maretzki, A.; dela Cruz, A.; Nickell, L. G.

    1971-01-01

    Evidence is presented for the increased excretion of amylolytic enzymes into a sugarcane cell culture medium when starch was substituted for sucrose as an energy source. The excretion was further enhanced by the inclusion of 1 μm gibberellic acid in the nutrient medium. The growth rate of the cells increased after they became adapted to starch relative to cells grown on sucrose, but the rate of amylolytic enzyme excretion remained unaltered. Amylolytic enzymes in the medium included α-amylase but the identity of one or more other enzymes related to starch hydrolysis remains in doubt. PMID:16657831

  18. Spatial distribution of niche and stem cells in ex vivo human limbal cultures.

    Science.gov (United States)

    Mariappan, Indumathi; Kacham, Santhosh; Purushotham, Jyothi; Maddileti, Savitri; Siamwala, Jamila; Sangwan, Virender Singh

    2014-11-01

    Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.

  19. 中药黄芩的悬浮培养及过氧化物同工酶酶谱分析%Analysis of scutellaria root suspension culture nutrient fluid and peroxidase isoenzyme

    Institute of Scientific and Technical Information of China (English)

    赵静; 张东向; 焦战战; 张令昂; 毕宇

    2014-01-01

    Scutellaria suspension cells as experimental materials,research of baicalin in suspension culture of suspension culture solution pH value,conductivity value,the concentration of sucrose,and different culture time determination of peroxide isozyme analysis,determination and cultivation of secondary metabolites of baicalin liquid and isoenzyme. In the baikal skullcap root suspension culture cycle,positive change and accumulation of secondary metabolites of baicalin spectrum peroxidase enzyme,14 d peroxidase activity of scutellaria baicalensis georgi strongest suspension culture,suspension culture of the 15 d when the highest concentration of baicalin.Suspension culture solution pH value,conductivity value,sucrose concentration of baicalin accumulation corresponding changes.%以黄芩悬浮细胞为实验材料,研究黄芩悬浮培养过程中悬浮培养液 pH 值、电导率值、蔗糖质量分数,并对不同培养时间测定过氧化物同工酶酶谱进行分析,确定次生代谢产物黄芩苷与培养液及同工酶的关系。结果表明,在黄芩悬浮培养周期中,过氧化物酶酶谱变化与次生代谢产物黄芩苷的积累呈正相关,黄芩悬浮培养14 d过氧化物酶活性最强,黄芩悬浮培养第15天时,黄芩苷质量浓度最高。悬浮培养液pH值、电导率值、蔗糖质量分数随黄芩苷累积均有相应变化。

  20. Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl.

    Science.gov (United States)

    Hoshino, Y; Nakano, M; Mii, M

    1995-03-01

    Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l(-1) 2,4-D and 2 g l(-1) casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1-3×10(7)/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l(-1) 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10(5)/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l(-1) each of NAA and BA for 2 months followed by 0.01 mg l(-1) NAA and 5 mg l(-1) BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant. PMID:24185329

  1. Preparation of Cell Cultures and Vaccinia Virus Stocks.

    Science.gov (United States)

    Cotter, Catherine A; Earl, Patricia L; Wyatt, Linda S; Moss, Bernard

    2015-01-01

    The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included.

  2. Isolation, suspension culture of the tumor cells from the malignant pleural effusion (MPE) of human lung adenocarcinoma and its identification%肺腺癌胸水肿瘤细胞分离与体外悬浮培养及鉴定

    Institute of Scientific and Technical Information of China (English)

    颜秉阳; 殷国强; 张志培; 赵亚超; 李小飞; 王小平

    2012-01-01

    Objective:Simulating the environment of MPE,to explore an appropriate method for the isolation and purication of the tumor cell from MPE and determine its specific gravity; and also to find a suitable dentify of cell and nutritive medium of cell suspension culture. Methods:MPE samples (1000ml each one) from 30 lung adenocarcino-ma with MPE patients who were histologically diagnosed. PRMI 1640 nutritive medium was used to suspense precipita-tion after centrifuged the 1000ml MPE, then, according to the 2 different isolation methods these samples were divided into 2 groups A and B ( n = 10). The tumor cells of the sample of group A were separated by discontinuous percoll density gradient centrifugation in one time by using 40%/20% percoll which specific gravity was 1. 056/1. 031. The tumor cells of the sample of group B were separated by density gradient centrifugation by using the human lymphocyte separation medium ( Ficoll - Hypaque ) which specific gravity was 1. 077 for the first time, and suspension cultivated in the PRMI 1640 medium with 10% fetal bovine serum (FBS) for48 hours,meanwhile,the tumor oells would gather into a mass, then, separated the tumor cells again by using the same isolation method of group A for the second time. Results;The yield rate of tumor cells of group A samples was lower then group B. Between the 30 samples of group A, there were only 6 sample yield a satisfy cell counts and primary cultured, 17 samples failed to yield and 7 samples got few cells. By contrast,in Group B, there were 23 sample yield a satisfy cell counts and primary culture,and 7 sam-ples failed to yield. The specific gravity of the tumor cell from MPE which was about 1. 050..Conclusion;The tumor cell from MPE can be effectively separated and purified by discontinuous density gradient centrifugation for two times. After centrifugation, the lymphocyte, monocyte and dendritic cells were centrifuged settling at the bottom of the centri-fuge tube,while the tumor cells were

  3. Transplantation of autologous noncultured epidermal cell suspension in treatment of patients with stable vitiligo

    Institute of Scientific and Technical Information of China (English)

    XU Ai-e; WEI Xiao-dong; CHENG Dong-qing; ZHOU He-fen; QIAN Guo-pei

    2005-01-01

    @@ Treatment of vitiligo by transplantation of noncultured melanocytes containing keratino-cytes has been successful since 1992,1 We report the encouraging results of autologous epidermal cell suspension in the treatment of 24 patients with stable vitiligo since 1998.

  4. OPTIMIZATION OF A MICROFLUIDIC DEVICE FOR DIFFUSION-BASED EXTRACTION OF DMSO FROM A CELL SUSPENSION

    OpenAIRE

    Fleming Glass, K. K.; Longmire, E. K.; Hubel, A.

    2008-01-01

    This study considers the use of a two-stream microfluidic device for extraction of dimethyl sulphoxide (DMSO) from a cryopreserved cell suspension. The DMSO diffuses from a cell suspension stream into a neighboring wash stream flowing in parallel. The model of Fleming et al.[14] is employed to determine and discuss optimal geometry and operating conditions for a case requiring removal of 95% DMSO from suspension streams with volumetric flow rates up to 2.5 ml/min. The effects of Peclet number...

  5. Effects of flame made zinc oxide particles in human lung cells - a comparison of aerosol and suspension exposures

    Directory of Open Access Journals (Sweden)

    Raemy David O

    2012-08-01

    Full Text Available Abstract Background Predominantly, studies of nanoparticle (NPs toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach however, does not reflect particle inhalation. As a more realistic simulation of such a scenario, efforts were made towards direct delivery of aerosols to air-liquid-interface cultivated cell cultures by the use of aerosol exposure systems. This study aims to provide a direct comparison of the effects of zinc oxide (ZnO NPs when delivered as either an aerosol, or in suspension to a triple cell co-culture model of the epithelial airway barrier. To ensure dose–equivalence, ZnO-deposition was determined in each exposure scenario by atomic absorption spectroscopy. Biological endpoints being investigated after 4 or 24h incubation include cytotoxicity, total reduced glutathione, induction of antioxidative genes such as heme-oxygenase 1 (HO–1 as well as the release of the (pro-inflammatory cytokine TNFα. Results Off-gases released as by-product of flame ZnO synthesis caused a significant decrease of total reduced GSH and induced further the release of the cytokine TNFα, demonstrating the influence of the gas phase on aerosol toxicology. No direct effects could be attributed to ZnO particles. By performing suspension exposure to avoid the factor “flame-gases”, particle specific effects become apparent. Other parameters such as LDH and HO–1 were not influenced by gaseous compounds: Following aerosol exposure, LDH levels appeared elevated at both timepoints and the HO–1 transcript correlated positively with deposited ZnO-dose. Under submerged conditions, the HO–1 induction scheme deviated for 4 and 24h and increased extracellular LDH was found following 24h exposure. Conclusion In the current study, aerosol and suspension-exposure has been compared by exposing cell cultures to equivalent amounts of ZnO. Both exposure strategies differ fundamentally in their

  6. 真菌诱导子对白桦悬浮体系中N和P的吸收利用和三萜合成的影响%Effect of fungal elicitors on N and P utilization and triterpene synthesis of Betula platyphylla suspension cell culture system

    Institute of Scientific and Technical Information of China (English)

    王晓东; 李晓灿; 翟俏丽; 詹亚光; 范桂枝

    2011-01-01

    Objective To analyze the relationship of N and P utilization and triterpene synthesis in birch (Betula platyphylla) suspension cell culture system induced by fungal elicitor. Methods Fungal elicitors (40 ug/mL) were added to the 8-day-old birch suspension cell culture, the change of N and P and triterpene content were analyzed by chemical colorimetry. Results The dry weight, triterpene content, and triterpene yield of birch cell decreased with the extension of fungal treatment time. Among them, triterpene content and yield were the highest after 1 d fungal induction, they were 21.98 mg/g and 123.82 g/L, respectively, about two times of the control. The pH value and conductivity of birch cell culture also reached a peak after 1 d fungal treatment with increase by 6.10% and 8.20%, respectively. The contents of nitrate, ammonium, and phosphate increased after fungal treatment, but the content of phosphate was reduced after day 1 and 2 induction by 28.67% and 15.68%. The correlations between phosphate, ammonium, nitrate and triterpenoid contents in birch cell medium were improved significantly after fungal treatment. Conclusion It is reasoned that increased synthesis of triterpenes after fungal elicitors inducement may be related to the concentration changes of phosphate, ammonium, and nitrate.%目的 分析真菌诱导的白桦悬浮体系中N、P的吸收利用和三萜合成的关系.方法 在白桦悬浮细胞的生长末期添加40 μg/mL真菌诱导子,采用比色法分析真菌诱导后白桦悬浮体系中N、P和三萜量的变化.结果 白桦细胞的干质量、三萜量和产量随着真菌处理时间的延长表现为逐渐降低趋势.其中,三萜量和产量均于真菌诱导后第1天达到最高,分别为21.98 mg/g和123.82 g/L,约为对照的2倍;白桦悬浮体系的pH值和电导率在真菌诱导后第1天达到高峰,pH值增长了6.10%,电导率增加了8.20%;除细胞内磷酸根质量分数在诱导的第1、2天分别降低了28

  7. Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures

    OpenAIRE

    Rival, A.; Ilbert, P.; Labeyrie, A.; E. Torres; Doulbeau, Sylvie; Personne, A.; Dussert, Stéphane; Beulé, T.; Durand-Gasselin, T.; Tregear, James; Jaligot, E.

    2013-01-01

    The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities. In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then pro...

  8. [Effect of calcium on medium alkalinization induced by salicylic acid in Salvia miltiorrhiza suspension cultures].

    Science.gov (United States)

    Liu, Liancheng; Wang, Cong; Dong, Juan'e; Su, Hui; Zhuo, Zequn; Xue, Yaxin

    2013-07-01

    We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.

  9. 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.

  10. Multivariate analyses of salt stress and metabolite sensing in auto- and heterotroph Chenopodium cell suspensions.

    Science.gov (United States)

    Wongchai, C; Chaidee, A; Pfeiffer, W

    2012-01-01

    Global warming increases plant salt stress via evaporation after irrigation, but how plant cells sense salt stress remains unknown. Here, we searched for correlation-based targets of salt stress sensing in Chenopodium rubrum cell suspension cultures. We proposed a linkage between the sensing of salt stress and the sensing of distinct metabolites. Consequently, we analysed various extracellular pH signals in autotroph and heterotroph cell suspensions. Our search included signals after 52 treatments: salt and osmotic stress, ion channel inhibitors (amiloride, quinidine), salt-sensing modulators (proline), amino acids, carboxylic acids and regulators (salicylic acid, 2,4-dichlorphenoxyacetic acid). Multivariate analyses revealed hirarchical clusters of signals and five principal components of extracellular proton flux. The principal component correlated with salt stress was an antagonism of γ-aminobutyric and salicylic acid, confirming involvement of acid-sensing ion channels (ASICs) in salt stress sensing. Proline, short non-substituted mono-carboxylic acids (C2-C6), lactic acid and amiloride characterised the four uncorrelated principal components of proton flux. The proline-associated principal component included an antagonism of 2,4-dichlorphenoxyacetic acid and a set of amino acids (hydrophobic, polar, acidic, basic). The five principal components captured 100% of variance of extracellular proton flux. Thus, a bias-free, functional high-throughput screening was established to extract new clusters of response elements and potential signalling pathways, and to serve as a core for quantitative meta-analysis in plant biology. The eigenvectors reorient research, associating proline with development instead of salt stress, and the proof of existence of multiple components of proton flux can help to resolve controversy about the acid growth theory. PMID:21974771

  11. 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

  12. Salt tolerance in cultured cells of Spartina pectinata.

    Science.gov (United States)

    Warren, R S; Baird, L M; Thompson, A K

    1985-04-01

    Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by both measures, was reduced in a roughly linear fashion between 0 and 500 mM NaCl. The pattern of growth in relation to NaCl is very similar to that of previously described cell suspensions derived from another halophyte, Distichlis spicata. In the field the latter is much more salt tolerant. The basis for the whole plant differences is not clear. They do not appear to reflect effectiveness of cell based salt tolerance or the presence of salt glands, which are reported here for the first time in S. pectinata and are found on the leaves of both species.

  13. A resonant structure designed for probing the elastic properties of suspension and adherent cells in liquid environments

    International Nuclear Information System (INIS)

    This paper presents a novel force sensitive structure exploiting a dynamic mode for probing the elastic properties of living cells. A key feature of this structure is the possibility of conducting measurements in liquid environments while keeping high dynamic performances. The structure indeed provides a steady area that can be adapted so that suspension or adherent cells can be placed in a culture medium. The steady area is also connected to two adjacent beam resonators. Because these resonators never need to be immersed into the culture medium during measurements, forces applied to cells can be estimated with a high sensitivity via frequency shifts. In this paper, we conduct an extensive theoretical analysis to investigate the nonlinear effects of large static pre-deflections on the dynamic behavior of the structure. As a proof of concept, we also report the fabrication, characterization and calibration of the first prototype intended to deal with suspension cells with a diameter ranging from 100 to 500 μm. This prototype currently offers a quality factor of 700 and a force sensitivity of ∼2.6 Hz mN−1. We also demonstrate that the prototype is capable of measuring the elastic modulus of biological samples in a rapid and sufficiently accurate manner without the need of a descriptive model. (paper)

  14. PREPARATION AND THE CULTURE OF LO2 CELLS ON PVA-BASED MICROCARRIERS

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong; YU Yaoting; PAN Jilun; WANG lianyong

    2001-01-01

    Using polyvinyl alcohol (PVA) as raw material and vacuum pump oil as oil phase medium,PVA-based microcarriers were prepared by suspension method The diameters of the beads were 100-180 μ m. LO2 cells were cultured on PVA-based microcarriers and cytodexⅢ microcarriers.Morphology, attachment and growth rate of LO2 cells were studied.

  15. PREPARATION AND THE CULTURE OF LO2 CELLS ON PVA—BASED MICROCARRIERS

    Institute of Scientific and Technical Information of China (English)

    ZHANGHong; YUYaoting; 等

    2001-01-01

    Using polyvinyl alcohol(PVA) as raw material and vacuum pump oil as oil phase medium,PVA-based microcarriers were prepared by suspension method,The diameters of the beads were 100-180um,LO2 cells were cultured on PVA-based microcarriers and cytodexⅢ microcarriers.Morphology,attachment and growth rate of LO2 cells were studied.

  16. Aseptic technique for cell culture.

    Science.gov (United States)

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here. PMID:18228291

  17. 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

  18. 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.

  19. 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.

  20. ON THE IMPROVEMENT OF THE PODOPHYLLOTOXIN PRODUCTION BY PHENYLPROPANOID PRECURSOR FEEDING TO CELL-CULTURES OF PODOPHYLLUM-HEXANDRUM ROYLE

    NARCIS (Netherlands)

    VANUDEN, W; PRAS, N; MALINGRE, TM

    1990-01-01

    In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferi

  1. 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.

  2. 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.

  3. 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. PMID:25940589

  4. Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua.

    Science.gov (United States)

    Baldi, Ashish; Dixit, V K

    2008-07-01

    Artemisinin, isolated from the shrub-Artemisia annua, is a sesquiterpene lactone used to treat multi-drug resistant strains of falciparum malaria. It is also effective against a wide variety of cancers such as leukemia and colon cancer. To counter the present low content in leaves and uneconomical chemical synthesis, alternate ways to produce artemisinin have been sought. But this compound remains elusive in cell cultures of A. annua despite the extensive studies undertaken. This work reports the first successful approach for production of artemisinin by cell cultures of Indian variety of A. annua. In the present study, an integrated yield enhancement strategy, developed by addition of selected precursor (mevalonic acid lactone) and elicitor (methyl jasmonate) at optimized concentrations, resulted in 15.2g/l biomass and 110.2mg/l artemisinin, which was 5.93 times higher in productivity in comparison to control cultures. PMID:17804216

  5. Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases

    NARCIS (Netherlands)

    Lens, P.N.L.; Gastesi, R.; Lettinga, G.

    2003-01-01

    This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell s

  6. 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.

  7. Conjugation of the mycotoxins alternariol and alternariol monomethyl ether in tobacco suspension cells.

    Science.gov (United States)

    Hildebrand, Andreas A; Kohn, Beate N; Pfeiffer, Erika; Wefers, Daniel; Metzler, Manfred; Bunzel, Mirko

    2015-05-20

    The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as β-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

  8. Instability of anthocyanin composition under different subculture conditions during long-term suspension cultures of Vitis vinifera L. var. Gamay Fréaux.

    Science.gov (United States)

    Qu, Junge; Zhang, Wei; Yu, Xingju

    2011-11-01

    The instability of secondary metabolite production is a ubiquitous problem in plant cell culture. In order to understand the instability in plant cell culture, we investigated anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, in our laboratory. Not only the anthocyanin contents but also its composition exhibited instability along with the long-term subculture. New methods were developed to indicate the instability of plant cell culture. Both the definition of instability coefficient (delta) and the application of factor scores were the first time in this field. To examine the effects of culture conditions on instability of anthocyanin biosynthesis, different subculture cycles and inoculum sizes had been investigated. Subculture cycle and inoculum size were both environmental cues driving the instability. Compared with subculture cycle, inoculum size was more effective in working on the instability of anthocyanin accumulation. Among all the conditions investigated in our study, (6.5 d, 2.00 g), (7 d, 2.00 g), (7.5 d, 2.00 g), (7 d, 1.60 g) and (7 d, 2.40 g), the condition of 7 d-subculture cycle together with 1.60 g-inoculum size was the best one to keep the stable production of anthocyanins. PMID:22393716

  9. Instability of anthocyanin composition under different subculture conditions during long-term suspension cultures of Vitis vinifera L. var. Gamay Fréaux.

    Science.gov (United States)

    Qu, Junge; Zhang, Wei; Yu, Xingju

    2011-11-01

    The instability of secondary metabolite production is a ubiquitous problem in plant cell culture. In order to understand the instability in plant cell culture, we investigated anthocyanin accumulation in suspension cultures of Vitis vinifera, as a model system, in our laboratory. Not only the anthocyanin contents but also its composition exhibited instability along with the long-term subculture. New methods were developed to indicate the instability of plant cell culture. Both the definition of instability coefficient (delta) and the application of factor scores were the first time in this field. To examine the effects of culture conditions on instability of anthocyanin biosynthesis, different subculture cycles and inoculum sizes had been investigated. Subculture cycle and inoculum size were both environmental cues driving the instability. Compared with subculture cycle, inoculum size was more effective in working on the instability of anthocyanin accumulation. Among all the conditions investigated in our study, (6.5 d, 2.00 g), (7 d, 2.00 g), (7.5 d, 2.00 g), (7 d, 1.60 g) and (7 d, 2.40 g), the condition of 7 d-subculture cycle together with 1.60 g-inoculum size was the best one to keep the stable production of anthocyanins.

  10. Insect Cell Culture and Biotechnology

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Begum, Parvin, E-mail: parvinchy@ees.hokudai.ac.jp; Fugetsu, Bunshi

    2013-09-15

    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.

  12. 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

  13. Efficient Electroporation of DNA and Protein into Confluent and Differentiated Epithelial Cells in Culture

    OpenAIRE

    Deora, Ami A.; Diaz, Fernando; Schreiner, Ryan; Rodriguez-Boulan, Enrique

    2007-01-01

    Electroporation-mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well-established procedures to electroporate polarized epithelial cells adhering to ...

  14. 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

    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...... 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...

  15. Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate.

    Science.gov (United States)

    Venereo-Sanchez, Alina; Gilbert, Renald; Simoneau, Melanie; Caron, Antoine; Chahal, Parminder; Chen, Wangxue; Ansorge, Sven; Li, Xuguang; Henry, Olivier; Kamen, Amine

    2016-06-17

    Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization

  16. Effect of Plant Growth Regulators on Callus, Cell Suspension and Cell Line Selection for Flavonoid Production from Pegaga (centella asiatica L. urban

    Directory of Open Access Journals (Sweden)

    Suat H. Tan

    2010-01-01

    Full Text Available Problem statement: Considering pegaga medicinal properties and over-exploitation, the requirement for a tissue culture technique as an alternative production system was crucial. Approach: Investigation of cell suspension culture response to different plant growth regulators (PRGs for flavonoid production from elite cell line was carried out. Callus cultures were initiated from the leaf explants of Centella asiatica on Murashige and Skoog (MS medium containing B5 vitamins and 30 g L−1 sucrose supplemented with different concentrations (0.5-2.5 mg L−1 of 2,4-D, NAA, Dicamba, Picloram and IBA supplied singly and in combination with different concentrations (0.5-1.5 mg L−1 of kinetin, BAP and TDZ. Results: Callus induction was observed for all the PGRs tested. The highest callus induction frequency (86.67% was observed in MS medium containing 2.0 mg L−1 2,4-D while the combination of 2.0 mg L−1 2,4-D and 1 mg L−1 kinetin in MS medium gave the highest biomass yield (0.27 g dry weight culture−1. This combination was also found to be best for callus proliferation for all the accessions investigated. Among the four accessions tested, UPM03 was found to have the highest biomass yield (0.041 g DW culture−1 and hydrolysed flavonoid content (10.75 mg g−1 DW after the 12th day of culture. The flavonoids present in the four accessions were quercetin, kaempherol, luteolin and rutin based on high performance liquid chromatography (HPLC analysis. These results indicated that C. asiatica accession UPM03 was the potential elite cell line in mass production of flavonoid, especially luteolin. Coclusions/Recommendations: In the establishment of cell suspension culture, 2 mg L−1 2,4-D and 1 mg L−1 kinetin were the best PGRs in supporting the cell growth and flavonoid production. This is the first report on the use of PRGs on the establishment of cell suspension cultures in flavonoid production of C. asiatica.

  17. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ecay, T.W.; Valentich, J.D. (Univ. of Texas Medical School, Houston (USA))

    1991-03-01

    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 {sup 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.

  19. Biotransformation of 14-Deacetoxy-13-oxo sinenxan A by Ginkgo Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    Jun Gui DAI; Meng ZHANG; Min YE; Wei Hua ZHU; Ji Yu GUO; Xiao Tian LIANG

    2003-01-01

    14-Deacetoxy-13-oxo sinenxan A (1) was converted to 9α-hydroxy-13-oxo-2α, 5α, 10β-triacetoxy-4(20),11-taxadiene (2) and 10β-hydroxy-13-oxo-2α,5α,9α-triacetoxy- 4(20), 11- taxadiene (3) by Ginkgo cell suspension cultures in 45% and 15% yields, respectively.

  20. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    Science.gov (United States)

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent.

  1. Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

    Science.gov (United States)

    Dores, C; Rancourt, D; Dobrinski, I

    2015-05-01

    To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

  2. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

    Yaping Feng; Hui Zhu; Jiang Hao; Xinmin Wang; Shengping Wu; Li Bai; Xiangming Li; Yun Zha

    2009-01-01

    BACKGROUND:Previous studies have used many methods for in vitro Schwann cells (SCs) cul-tures and purification,such as single cell suspension and cytosine arabinoside.However,it has been difficult to obtain sufficient cellular density,and the procedures have been quite tedious.OBJECTIVE:To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants.DESIGN,TIME AND SETTING:Cell culture and immunohistochemistry were performed at the Cen-tral Laboratory of Kunming General Hospital of Chinese PLA between March 2001 and October 2008.MATERIALS:Culture media containing 10% fetal bovine serum,as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco,USA;mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Bi-ological Products,China.METHODS:Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fe-tuses at 4-6 months pregnancy.Following removal of the dorsal root ganglion perineurium,the gan-glia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1),then explanted and cultured.SC purification was performed with 5 mL 10% fetal bovine serum added to the culture media,followed by differential adhesion.MAIN OUTCOME MEASURES:SCs morphology was observed under inverted phase contrast light microscopy.SC purity was evaluated according to percentage of S-100 immunostained cells.RESULTS:SCs were primarily cultured for 5-6 days and then subcultured for 4-5 passages.The highly enriched SC population reached > 95% purity and presented with normal morphology.CONCLUSION:A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

  3. 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.

  4. Polyamines in relation to growth in carrot cell cultures.

    Science.gov (United States)

    Fallon, K M; Phillips, R

    1988-09-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed.

  5. Polyamines in Relation to Growth in Carrot Cell Cultures 1

    Science.gov (United States)

    Fallon, Kevin M.; Phillips, Richard

    1988-01-01

    Changes in polyamine metabolism were investigated in relation to growth of cell suspension cultures of carrot (Daucus carota, cv Chantenay). Changes in levels of the major amines putrescine and spermidine throughout the culture period correlated poorly with changes in fresh weight, but a closer correlation with the minor component spermine was observed. The arginine decarboxylase (ADC) inhibitor difluoromethylarginine (DFMA) strongly and specifically inhibited ADC activity in the supernatant, reduced the major amine (putrescine) by 95% and the total amine content by 80%. It had no effect on cell number and stimulated fresh weight by over 25% through increased cell expansion. Spermine content, in contrast, increased with DFMA concentration in parallel with fresh weight increases. Difluoromethylornithine strongly inhibited ornithine decarboxylase activity in the pellet, but had little effect on either polyamine levels or culture growth. It was concluded that little evidence for a correlation between free polyamines and cell number in carrot cultures could be detected, but that a possible correlation between spermine content and cell expansion was observed. PMID:16666271

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

    International Nuclear Information System (INIS)

    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, 14C-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 [1-14C] acetic acid, and uniformly labelled lipids have been isolated from cultures that had been incubated with a mixture of [1-14C] acetic acid plus [2-14C] 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.)

  7. Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b6/f Complex

    Institute of Scientific and Technical Information of China (English)

    张贵友; 李萍; 朱瑞宇; 田瑞华; 戴尧仁

    2004-01-01

    An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco suspension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei,evidenced by DNA electrophoresis and fluorescence microscopy observations.Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still exhibited the apoptosis-inducing activity,but the supernatant had no effect,which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble.The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO,the specific inhibitor of Caspase-3,implying involvement of a Caspase-3-like protease during the process.Furthermore,extensive apoptosis in nuclei was induced by cytochrome b6/f on the thylakoid membrane,indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.

  8. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

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

  9. Dynamized preparations in cell culture.

    Science.gov (United States)

    Sunila, Ellanzhiyil Surendran; Kuttan, Ramadasan; Preethi, Korengath Chandran; Kuttan, Girija

    2009-06-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  10. Morphological characterization of cells in concentrated suspensions using multispectral diffuse optical tomography.

    Science.gov (United States)

    Hajihashemi, Mohammad Reza; Li, Xiaoqi; Jiang, Huabei

    2012-10-01

    Based on a non-spherical model of particle scattering, we investigate the capabilities and limitations of a T-matrix based inverse algorithm to morphologically characterize cells in concentrated suspensions. Here the cells are modeled as randomly orientated spheroidal particles with homogenous dielectric properties and suspended in turbid media. The inverse algorithm retrieves the geometrical parameters and the concentration of cells simultaneously by inverting the reduced scattering coefficient spectra obtained from multispectral diffuse optical tomography (MS-DOT). Both round and spheroidal cells are tested and the role of multiple and higher order scattering of particles on the performance of the algorithm is evaluated using different concentrations of cells.

  11. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

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

  12. Phase-segregated model for plant cell culture: The effect of cell volume fraction

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, W. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering]|[Tokyo Univ. (Japan)hinese Academy of Sciences, Dalian (China). Dalian Inst. of Chemical Physics; Furusaki, S. [Tokyo Univ. (Japan)] Middelberg, A. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering

    1998-06-01

    Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases. Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus. Large errors in the simulated results were observed if the phase-segregation was not considered. 12 refs., 3 figs.

  13. 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.

  14. The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

    Science.gov (United States)

    Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

    2016-01-15

    In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats.

  15. Establishing Embryogenic Callus Suspension Culture System in Black Locust%刺槐胚性细胞悬浮体系的建立1)

    Institute of Scientific and Technical Information of China (English)

    冯玥; 习洋; 陈串; 喻娃亚雄; 王少明; 徐惠敏; 孙宇涵; 李云

    2014-01-01

    以刺槐( Robinia pseudoacacia L.)未成熟合子胚为外植体材料,通过对外植体取材时期、基本培养基营养物质质量浓度、细胞起始密度、2,4-二氯苯氧乙酸(2,4-D)质量浓度等影响悬浮培养的因素的研究,建立了良好的刺槐胚性细胞悬浮系。研究结果表明:授粉后55 d的未成熟合子胚是为刺槐细胞悬浮培养提供胚性愈伤组织的最佳起始材料;添加有2.5 mg· L-1 NAA和0.5 mg· L-16-BA的1/2MS培养基为刺槐细胞悬浮培养的最适培养基;建立悬浮系的最适pH值范围在5.0~5.7;起始质量为2.0 g的悬浮系统其生长量增速较快,且细胞保持持续增长;当起始质量为3.0 g时,培养16 d后细胞进入缓慢增长期,且生长量有回落的趋势,起始质量为1.0 g时,细胞增殖速度非常缓慢,且容易出现褐化死亡的现象。%We employed embryogenic cell suspension ( ECS) induced from immature seeds of black locust ( Robinia pseudoaca-cia L.) as materials to study the effecting factors as explants period , nutrient concentration of basic medium , initial cell density and 2,4-D concentration of suspension culture medium to establish a successful embryogenic callus suspension cul -ture system for black locust.The seeds collected at eight weeks (about 55 days) post-anthesis obtained the highest ability to initiate embryogenic culture for embryogenic callus cell suspension establishment .The best callus initiation medium was 1/2MS+2.5 mg· L-1 NAA+0.5 mg· L-1 6-BA and the suitable pH ranged in 5.0-5.7.The cells maintained a sustained growth as initial density of suspension system was 2.0 g, the cells proliferation was slowed down after 16 days when the ini-tial density was 3.0 g, and the growth rate was with down trend .The cells were brown and died in slow proliferation rate with the initial density of 1.0 g.

  16. On the dielectric relaxation of biological cell suspensions: the effect of the membrane electrical conductivity.

    Science.gov (United States)

    Di Biasio, A; Cametti, C

    2011-06-01

    Due to the mismatch of the electrical parameters (the permittivity ϵ' and the electrical conductivity σ) of the membrane of a biological cell with the ones of the cytosol and the extracellular medium, biological cell suspensions are the site, under the influence of an external electric field, of large dielectric relaxations in the radiowave frequency range. However, a point still remains controversial, i.e., whether or not the value of membrane conductivity σ(s) might be extracted from the de-convolution of the dielectric spectra or otherwise if it would be more reasonable to assign to the membrane conductivity a value equal to zero. This point is not to be considered with superficiality since it concerns an a priori choice which ultimately influences the values of the electrical parameters deduced from this technique. As far as this point is concerned, the opinion of the researchers in this field diverges. We believe that, at least within certain limits, the membrane conductivity can be deduced from the shape of the relaxation spectra. We substantiate this thesis with two different examples concerning the first a suspension of human normal erythrocyte cells and the second a suspension of human lymphocyte cells. In both cases, by means of an accurate fitting procedure based on the Levenberg-Marquardt method for complex functions, we can evaluate the membrane conductivity σ(s) with its associated uncertainty. The knowledge of the membrane electrical conductivity will favor the investigation of different ion transport mechanisms across the cell membrane.

  17. "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. PMID:26971493

  18. Collision rates for rare cell capture in periodic obstacle arrays strongly depend on density of cell suspension.

    Science.gov (United States)

    Cimrák, I

    2016-11-01

    Recently, computational modelling has been successfully used for determination of collision rates for rare cell capture in periodic obstacle arrays. The models were based on particle advection simulations where the cells were advected according to velocity field computed from two dimensional Navier-Stokes equations. This approach may be used under the assumption of very dilute cell suspensions where no mutual cell collisions occur. We use the object-in-fluid framework to demonstrate that even with low cell-to-fluid ratio, the optimal geometry of the obstacle array significantly changes. We show computational simulations for ratios of 3.5, 6.9 and 10.4% determining the optimal geometry of the periodic obstacle arrays. It was already previously demonstrated that cells in periodic obstacle arrays follow trajectories in two modes: the colliding mode and the zig-zag mode. The colliding mode maximizes the cell-obstacle collision frequency. Our simulations reveal that for dilute suspensions and for suspensions with cell-to-fluid ratio 3.5%, there is a range of column shifts for which the cells follow colliding trajectories. However we showed, that for 6.9 and 10.4%, the cells never follow colliding trajectories. PMID:27023645

  19. Online flow cytometry for monitoring apoptosis in mammalian cell cultures as an application for process analytical technology.

    Science.gov (United States)

    Kuystermans, Darrin; Avesh, Mohd; Al-Rubeai, Mohamed

    2016-05-01

    Apoptosis is the main driver of cell death in bioreactor suspension cell cultures during the production of biopharmaceuticals from animal cell lines. It is known that apoptosis also has an effect on the quality and quantity of the expressed recombinant protein. This has raised the importance of studying apoptosis for implementing culture optimization strategies. The work here describes a novel approach to obtain near real time data on proportion of viable, early apoptotic, late apoptotic and necrotic cell populations in a suspension CHO culture using automated sample preparation in conjunction with flow cytometry. The resultant online flow cytometry data can track the progression of apoptotic events in culture, aligning with analogous manual methodologies and giving similar results. The obtained near-real time apoptosis data are a significant improvement in monitoring capabilities and can lead to improved control strategies and research data on complex biological systems in bioreactor cultures in both academic and industrial settings focused on process analytical technology applications.

  20. 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.

  1. A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

    Science.gov (United States)

    Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

    2016-02-01

    Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

  2. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

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

  3. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    Science.gov (United States)

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  4. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

    Science.gov (United States)

    Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

    2015-12-01

    Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

  5. Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures

    OpenAIRE

    Patil, Rohan A.; Kolewe, Martin E.; Normanly, Jennifer; Walker, Elsbeth L.; Roberts, Susan C.

    2012-01-01

    Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In this work, cul...

  6. In vitro culture of human thyroid cells

    International Nuclear Information System (INIS)

    Procedures for establishing primary cultures of human thyroid tissue are described. Tissues removed surgically from patients with papillary carcinoma (PC), follicular adenoma (FA), or hyperthyroidism were grown in culture. In addition, normal cells were separated from the margins of excised tumors and were also cultured. For each gram of thyroid tissue cultured, more than 1 x 105 cells attached to culture dishes. A mixture of 2.5 % fetal bovine serum supplemented with insulin, hydrocortisone, transferrin, glycl-1-histidyl-L-lysine acetate, somatostatin and epidermal growth factor was added to nutrient media containing equal parts of Ham's F-12 and minimum essential medium (αMEM). Complete medium selectively supported epithelial cell growth while restricting fibroblast cell growth, especially during the first two weeks of the primary culture. Cells were stimulated with thyroid stimulating hormone (TSH) and produced raised levels of cAMP and thyroid hormone (T3). Culture conditions that affected the response of cells to X-rays were identified. During the culture period, first and second passage cells were compared for differences in their radiosensitivities. In all cases, cells showed differences in their responses to radiation depending on the cell passage number. However, results of replicate experiments of first passage cells that were exposed to X-rays showed good agreement between experiments. This technique makes it possible to quantitate the effects of chemical and physical cytotoxic agents on proliferating human thyroid epithelial cells. (author)

  7. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

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

  8. 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.

  9. Development of a methodology for the propagation of 'Calcutta 4' (AA) and plantain genotypes from embryogenic cell suspensions and its interface with mutation breeding

    International Nuclear Information System (INIS)

    Bananas and plantains represent a major staple food for many millions of people in the tropics and subtropics. In Cuba, this crop constitutes a high priority of the national food program because of its capacity of producing fruit all year round, high demand and diversity of use, however, Black Sigatoka disease, caused by the leaf pathogen Mycosphaerella fijiensis, and pests and stress climatic conditions, has resulted in significant yield losses in this culture, which increases the production costs considerably. Somatic embryogenesis combined with mutation induction into in vitro culture mutations has not been studied in depth probably due to the low plant regeneration percentage of different Musa genotypes. Taking into consideration, the previous information following research objects are being developed: to develop a new methodology for developing somatic embryogenesis in different cultivar AAB, and cv. 'Calcutta 4', to develop a methodology for mutation induction with irradiations gamma rays to embryogenic cell suspensions and study anther culture of diploid cultivars. The genetic stability of plants obtained via embryogenesis through embryogenic cell suspensions showed the possibility to use the new methodology for developing somatic embryogenesis in different cultivar. From the results obtained for each cultivar, it is recommended to irradiate cv. 'Calcutta 4' at 50 Gy and cv. 'CEMSA 3/4' at 80 Gy. Irradiations with gamma rays to embryogenic cell suspensions resulted in a shorter stature in regenerated plants. Besides, plants of French plantain type were obtained from cultivar CEMSA 3/4. In Calcutta 4 the irradiation effect caused colour changes in plant pseudo-stems, and also deformed leaves were shown, but did not cause changes in the cultivar susceptibility to Black Sigatoka disease. In anther culture of diploid, callus induced from the state of uninuclate development was higher in relation to the other states studied and the regeneration plants are in

  10. Ghost Cell Suspensions as Blood Analogue Fluid for Macroscopic Particle Image Velocimetry Measurements.

    Science.gov (United States)

    Jansen, Sebastian V; Müller, Indra; Nachtsheim, Max; Schmitz-Rode, Thomas; Steinseifer, Ulrich

    2016-02-01

    Spatially resolved measurement of blood flow is of great interest in the development of artificial blood-carrying devices such as blood pumps, heart valve prostheses, and oxygenators. Particle image velocimetry (PIV) is able to measure instantaneous velocity fields in a plane with high accuracy and is being used more frequently for the development of such devices. However, as this measurement technique is based on optical access, blood flow at physiological hematocrit values is difficult to measure due to its low transparency and multiscattering properties. So far, only very small dimensions (in the range of 400 μm) can be measured using PIV. A suspension of ghost cells (GCs) offers a higher optical transparency than blood while having a similar rheological behavior. In this study, a procedure for the production of GC suspensions containing a very low intracellular hemoglobin concentration is presented. With the help of multiple rounds of controlled cell lysis, the intracellular hemoglobin concentration could be decreased to a point where a standard macroscopic PIV measurement was possible. A velocity profile of a 44% GC suspension in a circular channel with a diameter of 9.5 mm was measured with high spatial resolution. Meanwhile, the rheological behavior was found to be comparable with blood.

  11. Where the linearized Poisson-Boltzmann cell model fails: Spurious phase separation in charged colloidal suspensions

    Science.gov (United States)

    Tamashiro, M. N.; Schiessel, H.

    2003-07-01

    The Poisson-Boltzmann (PB) spherical Wigner-Seitz cell model—introduced to theoretically describe suspensions of spherical charged colloidal particles—is investigated at the nonlinear and linearized levels. The linearization of the mean-field PB functional yields linearized Debye-Hückel-type equations agreeing asymptotically with the nonlinear PB results in the weak-coupling (high-temperature) limit. Both the canonical (fixed number of microions) as well as the semigrand-canonical (in contact with an infinite salt reservoir) cases are considered and discussed in a unified linearized framework. In disagreement with the exact nonlinear PB solution inside a Wigner-Seitz cell, the linearized theory predicts the occurrence of a thermodynamical instability with an associated phase separation of the homogeneous suspension into dilute (gas) and dense (liquid) phases, being thus a spurious result of the linearization. We show that these artifacts, although thermodynamically consistent with quadratic expansions of the nonlinear functional and osmotic pressure, may be traced back to the nonfulfillment of the underlying assumptions of the linearization. This raises questions about the reliability of the prediction of gas/liquid-like phase separation in deionized aqueous suspensions of charged colloids mediated by monovalent counterions obtained by linearized theories.

  12. H_2O_2对水稻白叶枯病菌过氧化氢酶相关基因crg表达的诱导作用%Induction of bacterial catalase-related gene expression by H_2O_2 produced during interaction of rice suspension-cultured cells with Xanthomonas oryzae pv. oryzae or applied exogenously

    Institute of Scientific and Technical Information of China (English)

    周建波; 吴茂森; 胡俊; 何晨阳

    2009-01-01

    为了阐明H_2O_2对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)过氧化氢酶(CAT)相关基因(crg)表达的诱导作用,本研究定量分析了在水稻细胞-Xoo互作体系及其加入H_2O_2清除剂CAT后H_2O_2产量和crg表达;外源添加H_2O_2后的病菌生长和crg表达.结果表明:在互作条件下,H_2O_2含量稳定增加,10 h可达到峰值;在互作6 h时crg显著地被诱导表达;加入 CAT显著地降低了H_2O_2含量和crg表达;在外源H_2O_2胁迫条件下,H_2O_2以浓度效应的方式影响病菌增殖,显著地诱导了catB和srpA表达.因此,Xoo-水稻互作导致了H_2O_2的发生.无论是互作产生的还是外源的H_2O_2均显著地诱导了Xoo crg表达,从而活化了H_2O_2降解途径.%To elucidate the role of hydrogen peroxide (H_2O_2) produced during the interaction of rice suspension-cultured cells with Xanthomonas oryzae pv. oryzae (Xoo) or applied exogenously in inducing expression of bacterial catalase-related gene (crg), H_2O_2 production and crg expression during the rice-Xoo interaction, in which catalase (CAT) was exogenously added or not, were quantitatively analyzed. In vitro growth and crg expression of Xoo exposed to exogenously-applied H_2O_2 were quantitatively examined as well. Significant increase in H_2O_2 content and crg expression was observed during the interaction, while reduction in H_2O_2 concentration and crg expression was obviously found when CAT was exogenously added to the rice-Xoo interacting system. Growth in vitro was inhibited by exogenously-applied H_2O_2 in a dosage manner, which strongly induced the expression of catB and srpA. Therefore, H_2O_2 production was resulted from the rice-Xoo interaction, and crg expression was significantly induced by H_2O_2 either produced during the interaction or added exogenously.

  13. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    Science.gov (United States)

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  14. Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures.

    Science.gov (United States)

    Jackson, Jonathan P; Li, Linhou; Chamberlain, Erica D; Wang, Hongbing; Ferguson, Stephen S

    2016-09-01

    Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require time-consuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

  15. [Effects of beryllium chloride on cultured cells].

    Science.gov (United States)

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

    1984-05-01

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

  16. Production of L-DOPA by suspension grown cells of Mucuna pruriens.

    NARCIS (Netherlands)

    Wichers, H.J.

    1985-01-01

    In vitro cultured plant cells can be utilized for the production of valuable metabolites. Many biochemical and physiological characteristics of ln vitro cultured plant cells depend on various environmental parameters, such as the composition of the growth medium in which many parameters are represen

  17. Dynamic culture improves cell reprogramming efficiency.

    Science.gov (United States)

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

    2016-06-01

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

  18. 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

  19. 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.

  20. Autofluorescence of viable cultured mammalian cells.

    Science.gov (United States)

    Aubin, J E

    1979-01-01

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

  1. Frequency domain analysis of membrane capacitance of cultured cells (HeLa and myeloma) using the micropipette technique.

    OpenAIRE

    Asami, K; Takahashi, Y.; Takashima, S.

    1990-01-01

    The membrane capacitance and conductance of cultured cells (HeLa and mouse myeloma) are investigated using the micropipette method. Mean values of the membrane capacities were found to be 1.9 microF/cm2 for HeLa cells and 1.0 microF/cm2 for myeloma cells. These values are in agreement with those obtained using the suspension method. Whereas the suspension method is unable to provide the information on membrane conductance, the micropipette method is able to measure even an extremely small mem...

  2. Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells.

    Science.gov (United States)

    Mercx, Sébastien; Tollet, Jérémie; Magy, Bertrand; Navarre, Catherine; Boutry, Marc

    2016-01-01

    Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells. PMID:26870061

  3. 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.

  4. 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. PMID:26909780

  5. 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.

    2016-02-01

    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.

  6. Improved Biodegradation of 1,2,4-Trichlorobenzene by Adapted Microorganisms in Agricultural Soil and in Soil Suspension Cultures

    Institute of Scientific and Technical Information of China (English)

    SONG Yang; WANG Fang; F. O. KENGARA; BIAN Yong-Rong; YANG Xing-Lun; LIU Cui-Ying; JIANG Xin

    2011-01-01

    Inoculating soil with an adapted microbial community is a more effective bioaugrnentation approach than inoculation with pure strains in bioremediation.However,information on the potential of different inocula from sites with varying contamination levels and pollution histories in soil remediation is lacking.The objective of the study was to investigate the potential of adapted microorganisms in soil inocula,with different contamination levels and pollution histories,to degrade 1,2,4-trichlorobenzene (1,2,4-TCB).Three different soils from chlorobenzene-contaminated sites were inoculated into agricultural soils and soil suspension cultures spiked with 1,2,4-TCB.The results showed that 36.52% of the initially applied 1,2,4-TCB was present in the non-inoculated soil,whereas about 19.00% of 1,2,4-TCB was present in the agricultural soils inoculated with contaminated soils after 28 days of incubation.The soils inoculated with adapted microbial biomass (in the soil inocula) showed higher respiration and lower 1,2,4-TCB volatilization than the non-inoculated soils,suggesting the existence of 1,2,4-TCB adapted degraders in the contaminated soils used for inoculation.It was further confirmed in the contaminated soil suspension cultures that the concentration of inorganic chloride ions increased continuously over the entire experimental period.Higher contamination of the inocula led not only to higher degradation potential but also to higher residue formation.However,even inocula of low-level contamination were effective in enhancing the degradation of 1,2,4-TCB.Therefore,applying adapted microorganisms in the form of soil inocula,especially with lower contamination levels,could be an effective and environment-friendly strategy for soil remediation.

  7. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

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

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long......-term cultures. Support protocols describe methods for maintenance of vector-producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long-term cultures. Other protocols provide PCR...

  8. 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. PMID:24470393

  9. Methods for Maintaining Insect Cell Cultures

    OpenAIRE

    Lynn, Dwight E.

    2002-01-01

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains.

  10. A novel automated bioreactor for scalable process optimisation of haematopoietic stem cell culture.

    Science.gov (United States)

    Ratcliffe, E; Glen, K E; Workman, V L; Stacey, A J; Thomas, R J

    2012-10-31

    Proliferation and differentiation of haematopoietic stem cells (HSCs) from umbilical cord blood at large scale will potentially underpin production of a number of therapeutic cellular products in development, including erythrocytes and platelets. However, to achieve production processes that are scalable and optimised for cost and quality, scaled down development platforms that can define process parameter tolerances and consequent manufacturing controls are essential. We have demonstrated the potential of a new, automated, 24×15 mL replicate suspension bioreactor system, with online monitoring and control, to develop an HSC proliferation and differentiation process for erythroid committed cells (CD71(+), CD235a(+)). Cell proliferation was relatively robust to cell density and oxygen levels and reached up to 6 population doublings over 10 days. The maximum suspension culture density for a 48 h total media exchange protocol was established to be in the order of 10(7)cells/mL. This system will be valuable for the further HSC suspension culture cost reduction and optimisation necessary before the application of conventional stirred tank technology to scaled manufacture of HSC derived products.

  11. Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Linn

    Directory of Open Access Journals (Sweden)

    Vijai Singh Karwasara

    2011-01-01

    Full Text Available Background: Glycyrrhizin, obtained from Abrus precatorius (Indian liquorice, is a phytoconstituent of importance for pharmaceutical and food industries. Materials and Methods: High producing and fast growing cell lines of A. precatorius were developed by transformation with Agrobacterium tumefaciens for glycyrrhizin production. Its maximum transformation efficiency of 85% was obtained by infecting leaves with A. tumefaciens MTCC-431 supplemented with 50 μM acetosyringone. Thorough culture growth kinetics with sugar consumption profiles was established. Results: A twofold increase in glycyrrhizin productivity was obtained in transformed A. precatorius cell suspension cultures over the untransformed cultures. The fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer were tested at different concentrations to enhance glycyrrhizin production in transformed cell suspension cultures of A. precatorius. Maximum enhancement of 4.9- and 3.8-fold in glycyrrhizin contents, were obtained with A. niger (7.5% v/v and R. stolonifer (5.0% v/v, respectively, on the 5th day after elicitor treatment. Conclusion: This study indicates the prospective of the amalgamation of elicitation methodology with transformed cell cultures for the large-scale production of glycyrrhizin.

  12. Enhanced production of L-DOPA in cell cultures of Mucuna pruriens L. and Mucuna prurita H.

    Science.gov (United States)

    Raghavendra, S; Kumar, V; Ramesh, C K; Khan, M H Moinuddin

    2012-01-01

    A comparative study on the production of 3,4-dihydroxyphenylalanine (L-DOPA) was carried out in cell cultures of two Mucuna species by elicitor treatment and precursor feeding. The influence of elicitors and the precursor molecule on L-DOPA production, polyphenol oxidase (PPO) and tyrosinase activities was also studied. Callus cultures were initiated in Mucuna pruriens L. and Mucuna prurita H. on MS medium supplemented with BAP and IAA at different concentrations. Suspension cultures were established in MS liquid medium supplemented with BAP, IAA, the elicitors methyl jasmonate, chitin and pectin or the precursor L-tyrosine at different concentrations for L-DOPA production. Compared to the controls, several-fold increases in L-DOPA concentration were observed in elicitor-treated and precursor-fed suspension cultures of both plant species. L-DOPA concentrations were comparatively higher in precursor-fed cultures than those receiving elicitor treatments. A parallel increase in tyrosinase and PPO levels was also observed. Loss of cell viability was observed at high concentrations of elicitor-treated cultures, whereas L-tyrosine did not cause any cell death. Compared to elicitor treatments, precursor feeding resulted in higher concentrations of L-DOPA production and tyrosinase activity. The efficacy of L-DOPA production was found to be higher for suspension cultures of M. pruriens compared to M. prurita in all treatments. PMID:21995853

  13. Culture and transfection of axolotl cells.

    Science.gov (United States)

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

    2015-01-01

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

  14. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

    Science.gov (United States)

    Amer, Luke D; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J; Bryant, Stephanie J

    2015-08-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

  15. A simple eccentric stirred tank mini-bioreactor: mixing characterization and mammalian cell culture experiments.

    Science.gov (United States)

    Bulnes-Abundis, David; Carrillo-Cocom, Leydi M; Aráiz-Hernández, Diana; García-Ulloa, Alfonso; Granados-Pastor, Marisa; Sánchez-Arreola, Pamela B; Murugappan, Gayathree; Alvarez, Mario M

    2013-04-01

    In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5-100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple-easy to assemble, easy to use, easy to clean-cell culture mini-bioreactors for lab-scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini-bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini-bioreactor were comparable to those observed for 6-well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini-bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini-bioreactor.

  16. Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling

    Directory of Open Access Journals (Sweden)

    Emil Pásztor

    2011-08-01

    Full Text Available The morphology and hormone production of pituitary adenoma cell cultures were compared in order to highlight their characteristic in vitro features. Cell suspensions were prepared from 494 surgical specimens. The 319 viable monolayer cultures were analyzed in detail by light microscopy and immunocytochemistry within two weeks of cultivation. Some cultures were further characterized by scanning, transmission and immunogold electron microscopy. The viability and detailed in vitro morphology of adenoma cells were found to be characteristic for the various types of pituitary tumors. The sparsely granulated growth hormone, the corticotroph and the acidophil stem cell adenomas provided the highest ratio of viable cultures. Occasionally, prolonged maintenance of cells resulted in long-term cultures. Furthermore, a variety of particular distributions of different hormone-containing granules were found in several cases. Both light microscopic and ultrastructural analyses proved that the primary cultures of adenoma cells retain their physiological features during in vitro cultivations. Our in vitro findings correlated with the routine histopathological examination. These results prove that monolayer cultures of pituitary adenoma cells can contribute to the correct diagnosis and are valid model systems for various oncological and neuroendocrinological studies.

  17. Bioimpedance analysis for the characterization of breast cancer cells in suspension.

    Science.gov (United States)

    Guofeng Qiao; Wei Wang; Wei Duan; Fan Zheng; Sinclair, A J; Chatwin, C R

    2012-08-01

    The bioimpedance spectroscopy (BIS) technique is potentially a useful tool to differentiate malignancy based on the variation of electrical properties presented by different tissues and cells. The different tissues and cells present variant electrical resistance and reactance when excited at different frequencies. The main purpose of this area of research is to use impedance measurements over a low-frequency bandwidth ranging from 1 kHz to 3 MHz to 1) differentiate the pathological stages of cancer cells under laboratory conditions and 2) permit the extraction of electrical parameters related to cellular information for further analysis. This provides evidence to form the basis of bioimpedance measurement at the cellular level and aids the potential future development of rapid diagnostics from biopsy materials. Three cell lines, representing normal breast epithelia and different pathological stages of breast cancer, have been measured using a standard impedance analyzer driving a four-electrode chamber filled with different cell suspensions. We identify the specific BIS profile for each cell type and determine whether these can be differentiated. In addition, the electrical parameters, e.g., the intracellular conductivity, membrane capacitance/capacity, characteristic frequency, are extracted by the use of equivalent circuit models and physical models to provide details of the cell electric signatures for further analysis of cancer cell pathology. PMID:22692870

  18. A device to facilitate preparation of high-density neural cell cultures in MEAs.

    Science.gov (United States)

    Mok, S Y; Lim, Y M; Goh, S Y

    2009-05-15

    A device to facilitate high-density seeding of dissociated neural cells on planar multi-electrode arrays (MEAs) is presented in this paper. The device comprises a metal cover with two concentric cylinders-the outer cylinder fits tightly on to the external diameter of a MEA to hold it in place and an inner cylinder holds a central glass tube for introducing a cell suspension over the electrode area of the MEA. An O-ring is placed at the bottom of the inner cylinder and the glass tube to provide a fluid-tight seal between the glass tube and the MEA electrode surface. The volume of cell suspension in the glass tube is varied according to the desired plating density. After plating, the device can be lifted from the MEA without leaving any residue on the contact surface. The device has enabled us to increase and control the plating density of neural cell suspension with low viability, and to prepare successful primary cultures from cryopreserved neurons and glia. The cultures of cryopreserved dissociated cortical neurons that we have grown in this manner remained spontaneously active over months, exhibited stable development and similar network characteristics as reported by other researchers. PMID:19428539

  19. 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.

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  1. Cell culture models for study of differentiated adipose cells

    OpenAIRE

    Clynes, Martin

    2014-01-01

    Adipose cells are an important source of mesenchymal stem cells and are important for direct use in research on lipid metabolism and obesity. In addition to use of primary cultures, there is increasing interest in other sources of larger numbers of cells, using approaches including induced pluripotent stem cell differentiation and viral immortalisation.

  2. Trueness-to-type and agronomic characteristics of Coffea arabica trees micropropagated by the embryogenic cell suspension technique.

    Science.gov (United States)

    Etienne, H; Bertrand, B

    2001-09-01

    Trueness-to-type and agronomic characteristics of trees of four coffee (Coffea arabica L.) F(1) hybrid clones derived from embryogenic cell suspensions were compared with those of trees produced from in vitro microcuttings. Three types of variants were observed among the 644 trees derived from embryogenic suspensions. Total frequency of the variants was 2.1% for trees originating from embryogenic cell suspensions, whereas no variant was found among the trees produced from microcuttings. The variant known as "thick leaf" had thick leaves, many abnormally starry flowers and low yields of large fruit. The "dwarf" variant was characterized by slow growth and small fruit. The "dwarf peaberry" variant had abnormal seeds in a single cavity, in addition to the "thick leaf" and "dwarf" characteristics. Compared with normal trees, the variants differed in leaf density and number of chloroplasts per guard cell. The variants aside, there were no differences in the main agronomic characteristics between trees produced from embryogenic suspensions and those produced from microcuttings. For all four clones, the trees had vegetative characteristics, productivity, fertility, and bean biochemical, mineral and organoleptic characteristics that were identical to those of the controls. We conclude that it is possible to generate coffee trees commercially with normal agronomic performance from embryogenic suspensions, because the frequency with which somaclonal variants occur is limited.

  3. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    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 ZnC12, one being increased in amount while the other was reduced to an insignificant level. In ZnC12-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 ZnC12-treated cells. While ZnC12 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 ZnC12-treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC12-treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  4. 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 productivit

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

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R; Garbe, James C

    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.

  7. Cell culture monitoring by impedance mapping using a multielectrode scanning impedance spectroscopy system (CellMap)

    International Nuclear Information System (INIS)

    We report on the impedance mapping of in vitro cellular morphology by electrical impedance spectroscopy, using microelectrodes. A micro multielectrode system was designed, fabricated, assembled, tested and demonstrated for the monitoring of anchorage-dependent cell behavior and morphology. This system allowed continuous, label-free, quantitative monitoring and visualization of cell adhesion, spreading, proliferation and detachment due to cell cycle processes as well as cell–drug interaction, with spatio-temporal resolution. OvCa429 ovarian cancer cells were monitored in vitro over a period of 70 hours by inoculating the cell suspension directly on the multielectrode device. The phase angle of impedance was observed to develop a distinctive shape as a result of cell attachment and proliferation. The shape of the phase angle curve reverted back to the pre-attachment shape upon detachment of cells from the substrate, caused by the addition of trypsin to the cell culture medium. The impedance data of the cell culture were then successfully modeled as a multi-parametric equivalent circuit. The model incorporated both interfacial and cell-layer impedance parameters. Upon addition of trypsin, the cell-layer parameters showed a marked decline and were eventually eliminated from the multi-parametric model, confirming the correlation of the model to the electrode–cell–electrolyte system. These experiments demonstrate the applicability of the impedance mapping technique in visualizing and quantifying physiological changes in the cell layer due to cellular processes as well as the effect of external chemical stimulus on cells (cell–drug interaction)

  8. Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture 1

    Science.gov (United States)

    Maki, Hisae; Ando, Satoshi; Kodama, Hiroaki; Komamine, Atsushi

    1991-01-01

    Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G1 phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G1 to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs. PMID:16668290

  9. Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

    Science.gov (United States)

    Ledoux, Quentin; Van Cutsem, Pierre; Markό, Istvan E; Veys, Pascal

    2014-01-01

    H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

  10. 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...

  11. Simple spinner bottle with rotating basket packed with carriers for hybridoma cell culture.

    Science.gov (United States)

    Chen, Y; Wang, G; Zhang, W; Freedman, D

    1996-01-01

    r-69B is a mouse-mouse hybridoma cell line, producing monoclonal antibody IgG against human r-IFN. It was cultured for 21 days in the 1.0-L spinner bottle which was assembled with a rotating basket packed with the 8.0-g Fibra-Cel carriers. The agitation was 100 r/min. The results showed that 53.5% of the cells can be trapped within the carriers in the basket and the cell concentration and MAb was about double those in the suspension culture. The spinner bottle could be assembled simply and used in general laboratories. It also could be used for different kinds of cells, including anchorage-dependent and independent cells. PMID:9093764

  12. Fast filtration sampling protocol for mammalian suspension cells tailored for phosphometabolome profiling by capillary ion chromatography - tandem mass spectrometry.

    Science.gov (United States)

    Kvitvang, Hans F N; Bruheim, Per

    2015-08-15

    Capillary ion chromatography (capIC) is the premium separation technology for low molecular phosphometabolites and nucleotides in biological extracts. Removal of excessive amounts of salt during sample preparation stages is a prerequisite to enable high quality capIC separation in combination with reproducible and sensitive MS detection. Existing sampling protocols for mammalian cells used for GC-MS and LC-MS metabolic profiling can therefore not be directly applied to capIC separations. Here, the development of a fast filtration sampling protocol for mammalian suspension cells tailored for quantitative profiling of the phosphometabolome on capIC-MS/MS is presented. The whole procedure from sampling the culture to transfer of filter to quenching and extraction solution takes less than 10s. To prevent leakage it is critical that a low vacuum pressure is applied, and satisfactorily reproducibility was only obtained by usage of a vacuum pressure controlling device. A vacuum of 60mbar was optimal for filtration of multiple myeloma Jjn-3 cell cultures through 5μm polyvinylidene (PVDF) filters. A quick deionized water (DI-water) rinse step prior to extraction was tested, and significantly higher metabolite yields were obtained during capIC-MS/MS analyses in this extract compared to extracts prepared by saline and reduced saline (25%) washing steps only. In addition, chromatographic performance was dramatically improved. Thus, it was verified that a quick DI-water rinse is tolerated by the cells and can be included as the final stage during filtration. Over 30 metabolites were quantitated in JJN-3 cell extracts by using the optimized sampling protocol with subsequent capIC-MS/MS analysis, and up to 2 million cells can be used in a single filtration step for the chosen filter and vacuum pressure. The technical set-up is also highly advantageous for microbial metabolome filtration protocols after optimization of vacuum pressure and washing solutions, and the reduced salt

  13. 接种菌悬液培养方式对纺织品抗菌试验的影响%Effect of Inoculation Suspension Culture Method on Textile Antibacterial Test

    Institute of Scientific and Technical Information of China (English)

    张红利; 刘会; 蒋芳

    2011-01-01

    探讨纺织品抗茵性能检测中接种菌悬液的培养方式对抗菌试验的影响.分析了接种茵悬液浓度与试验有效性的关系,分别采用振荡培养和静止培养方式制取金黄色葡萄球茵和大肠杆菌悬液,通过对比所制得接种茵悬液的浓度,认为在接种量相同的情况下,采用静置培养所得的金黄色葡萄球菌的接种菌悬液浓度符合标准要求,而采用振荡培养所得的大肠杆菌的接种菌悬液符合标准要求.%Effect of inoculation suspension culture method in textile antibacterial test on antibacterial test was discussed. Relationship between inoculation suspension concentration and test effectiveness was analyzed, staphylococcus aureus suspension and bacillus coli suspension were cultured by oscillation culture and static culture respectively.Through contrasting the suspensions concentration,it is considered that in condition of same inoculum amount, staphylococcus aureus suspension concentration got in static culture could fit standard demand while bacillus coli suspension got by oscillation culture could fit standard demand.

  14. Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells

    Directory of Open Access Journals (Sweden)

    Bortesi Luisa

    2012-07-01

    Full Text Available Abstract Background Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10. Results We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5′-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. Conclusions We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.

  15. Pinoresinol from Ipomoea cairica cell cultures.

    Science.gov (United States)

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

    2002-10-01

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

  16. New Synthetic Pyridine Derivate as Potential Elicitor in Production of Isoflavonoids and Flavonoids in Trifolium pratense L. Suspension Culture

    Directory of Open Access Journals (Sweden)

    Marie Kašparová

    2012-01-01

    Full Text Available The production of secondary metabolites in Trifolium pratense L. suspension culture of the family of legume plants (Fabaceae is low, and therefore there was an attempt to increase it by elicitation. New synthetic substance, 2-(2-fluoro-6-nitrobenzylsulfanylpyridine-4-carbothioamide, was tested as elicitor—a substance that showed the best elicitation effect after 48-hour application of 1 μmol L−1 concentration. Maximum contents of genistin (11.60 mg g−1 DW, daidzein (8.31 mg g−1 DW, and genistein (1.50 mg g−1 DW were recorded, and the production of these isoflavonoids thus significantly increased, when compared with the control, by 152%, 151%, and 400%. The maximum content of flavonoids (5.78 mg g−1 DW and the increase in the production by 142%, when compared with the control, were induced by 6-hour application of 100 μmol L−1 concentration. The tested substance showed to be an effective elicitor of phenylpropane metabolism.

  17. 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.

  18. Embryogenic suspensions of adult cork oak: the first step towards mass propagation.

    OpenAIRE

    Jiménez García, Jesús; López Vela, Dolores; Ruiz Galea, Mar; Celestino, Cristina; Toribio, Mariano; Alegre, Jesús

    2013-01-01

    Abstract Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained ...

  19. Growth regulation of cultured human nevus cells.

    Science.gov (United States)

    Mancianti, M L; Györfi, T; Shih, I M; Valyi-Nagy, I; Levengood, G; Menssen, H D; Halpern, A C; Elder, D E; Herlyn, M

    1993-03-01

    Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo. PMID:8440904

  20. Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells

    OpenAIRE

    Lee, Jun Hee; Han, Yong-Seok; Lee, Sang Hun

    2016-01-01

    Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes in...

  1. Suicidal tomato cells : programmed cell death in suspension-cultured tomato cells and ripening fruit

    NARCIS (Netherlands)

    Hoeberichts, F.A.

    2002-01-01

     Tomato fruit ripening involves a series of highly organised biochemical, physiological and structural changes that are under strict genetic control. The plant hormone ethylene (C 2 H 4 ), in synergy with certain developmental cues, regulates fruit ripening b

  2. 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 ( 13C-NMR) were used to

  3. General overview of neuronal cell culture.

    Science.gov (United States)

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

    2013-01-01

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

  4. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways. PMID:27590152

  5. Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

    Institute of Scientific and Technical Information of China (English)

    Li Fei; Chengchuan Jiang; Linyin Feng; Yaodong Ji; Zhongliang Ding

    2006-01-01

    BACKGROUND: Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy (CRT) for treating Parkinson disease (PD). Embryonic mesencephalic precursor cells (MPCs) can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far.OBJECTIVE: To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats.DESIGN: A randomized and controlled trial taking SD rats as experimental animals.SETTING: Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS: This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats,with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences.METHODS: Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1out of 20 model rats after execution, and the other 19 rats were randomly divided into control group (n=5),sham transplantation group (n=5)and cell grafted group (n=9). Primary cultured E12 MPC cell suspension (1.2×1011 L-1)were used as donor cells. 4 μL primary cultured E12 MPC cell suspension prepared freshly was injected

  6. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

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

  7. Production and antigenic properties of influenza virus from suspension MDCK-siat7e cells in a bench-scale bioreactor.

    Science.gov (United States)

    Chu, Chia; Lugovtsev, Vladimir; Lewis, Andrew; Betenbaugh, Michael; Shiloach, Joseph

    2010-10-18

    In efforts to overcome limitations associated with egg-based influenza vaccines, mammalian cell substrates have gradually emerged as potential production platforms. Recently, a suspension Madin Darby canine kidney (MDCK) cell line for influenza virus production was created by expressing the human siat7e gene. To examine the broad susceptibility of this novel cell line, the scalability of the production process, and the antigenic stability of cell-derived progeny viruses, infection experiments using four current influenza vaccine strains (A/California/07/2009 X-179A H1N1, A/Brisbane/59/2007 IVR-148 H1N1, A/Uruguay/716/2007 X-175C H3N2, and B/Brisbane/60/2008) were performed. In small-scale experiments, this cell line was found to support high-titer replication of all four virus strains. Subsequently, production in a bench-scale bioreactor and the antigenic characteristics of progeny viruses were assessed. High titers of hemagglutinin (at least 1:512) were produced in a 2-L bench-scale bioreactor with all four strains. Immunoblot results demonstrated higher yields in the cells than those obtained in chicken embryonated eggs with three of the four tested strains. Progeny viruses collected after serial passages in this cell line exhibited minimal mutations in the HA-encoding gene. Hemagglutination inhibition (HAI) assays using ferret antiserum confirmed the antigenic stability. As a proof-of-concept this work demonstrates that by using a proper strategy, high yields of biologically active hemagglutinin can be produced from scalable cultures of suspension MDCK-siat7e cells. PMID:20800699

  8. Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams.

    Science.gov (United States)

    Wongsamuth, R; Doran, P M

    1994-08-01

    Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.

  9. Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams.

    Science.gov (United States)

    Wongsamuth, R; Doran, P M

    1994-08-01

    Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc. PMID:18618782

  10. Selective growth of freshly isolated human breast epithelial cells cultured at low concentrations in the presence or absence of bone marrow cells.

    Science.gov (United States)

    Emerman, J T; Stingl, J; Petersen, A; Shpall, E J; Eaves, C J

    1996-01-01

    In this study, we show that conditions previously found to promote the selective growth of human breast epithelial cells (HBEC) in serum-free primary cultures established from normal or malignant tissue can be extended to cultures initiated at low seeding densities (horse serum, which promotes the growth of many types of stromal cells and associated hematopoietic precursors, or by the inclusion in the initial cell suspension of marrow cells at HBEC to marrow cell ratios typical of bone marrow samples from patients with metastatic breast cancer. The presence of fibroblast feeders from a variety of sources enhanced the growth of HBEC to different degrees. In cultures initiated with low numbers of cells obtained from samples of breast carcinoma, HBEC growth was generally reduced by comparison to cultures of normal HBEC. With the detection methods used, it was not possible to determine the extent to which this decreased growth was due to a reduced frequency of malignant HBEC with in vitro precursor activity, or the presence of reduced numbers of residual normal HBEC precursors, or both. However, preliminary data indicate that this approach also allows the detection of some breast carcinoma cells with proliferative ability that are present in the marrow or pleural effusions of some breast cancer patients. These studies demonstrate the feasibility of detecting normal and malignant HBEC with growth potential when these are cultured at low density and/or as rare contaminants of marrow cell suspensions, and provide a starting point for their further characterization. PMID:8944333

  11. Biocompatibility Studies on Fibrin Glue Cultured with Bone Marrow Mesenchymal Stem Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    方煌; 彭松林; 陈安民; 黎逢峰; 任凯; 胡宁

    2004-01-01

    Summary: By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro,the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering.

  12. Integrated biosensors for cell culture monitoring

    OpenAIRE

    De Micheli, Giovanni; Boero, Cristina; Olivo, Jacopo; Carrara, Sandro

    2014-01-01

    Biosensors for endogenous compounds, such as glucose and lactate, are applied to monitor cell cultures. Cells can be cultivated for several purposes, such as understanding and modeling some biological mechanisms, the development of new drugs and therapies, and in the field of regenerative medicine. We have realized a self-contained monitoring system with remote readout. Metabolite detection is based on oxidases immobilized onto carbon nanotubes. We calibrate the system for glucose and lactate...

  13. Cell culture from sponges: pluripotency and immortality

    NARCIS (Netherlands)

    Caralt Bosch, de S.; Uriz, M.J.; Wijffels, R.H.

    2007-01-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a

  14. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

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

  15. 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

  16. Cell culture models using rat primary alveolar type I cells.

    Science.gov (United States)

    Downs, Charles A; Montgomery, David W; Merkle, Carrie J

    2011-10-01

    There is a lack of cell culture models using primary alveolar type I (AT I) cells. The purpose of this study was to develop cell culture models using rat AT I cells and microvascular endothelial cells from the lung (MVECL). Two types of model systems were developed: single and co-culture systems; additionally a 3-dimensional model system was developed. Pure AT I cell (96.3 ± 2.7%) and MVECL (97.9 ± 1.1%) preparations were used. AT I cell morphology, mitochondrial number and distribution, actin filament arrangement and number of apoptotic cells at confluence, and telomere attrition were characterized. AT I cells maintained their morphometric characteristics through at least population doubling (PD) 35, while demonstrating telomere attrition through at least PD 100. Furthermore, AT I cells maintained the expression of their specific markers, T1α and AQ-5, through PD 42. For the co-cultures, AT I cells were grown on the top and MVECL were grown on the bottom of fibronectin-coated 24-well Transwell Fluroblok™ filter inserts. Neither cell type transmigrated the 1 μm pores. Additionally, AT I cells were grown in a thick layer of Matrigel(®) to create a 3-dimensional model in which primary AT I cells form ring-like structures that resemble an alveolus. The development of these model systems offers the opportunities to investigate AT I cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  17. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M;

    2006-01-01

    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...

  18. Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

    OpenAIRE

    Szechynska-Hebda, M.; Wedzony, M.; Dubas, E.; Kieft, H; Lammeren, van, ACAP Andre

    2006-01-01

    Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzym...

  19. 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

  20. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

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

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our foc...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....... in this paper is on the optimization of how a biochemical application is performed on a biochip. In this paper, we consider cell culture biochips, where several cell colonies are exposed to soluble compounds and monitored in real-time to determine the right combination of factors that leads to the desired...

  1. Effect of corticosteroid binding proteins on the steroidogenic activity of bovine adrenocortical cell suspensions.

    Science.gov (United States)

    Basset, M; Rostaing-Metz, B; Chambaz, E M

    1982-07-01

    The possible role of steroid binding proteins in the hormonal secretion process of a steroidogenic tissue was examined using bovine adrenocortical cell suspensions, either under basal conditions or in the presence of half-maximally active concentration (1 x 10(-9) M) of synthetic adrenocorticotropic hormone (ACTH). Three types of plasma cortisol binding proteins were used, namely bovine serum albumine (BSA), purified transcortin (CBG) and purified anticortisol immunoglobulins (IgG). When added to the incubation medium, CBG (at 1 x 10(-10) to 2 x 10(-9) M cortisol binding sites) and anticortisol IgG (at 4.8 x 10(-10) to 3 x 10(-9) M cortisol binding sites) did not influence either the basal nor the ACTH-stimulated net cortisol production of the cell preparations. Whereas crystallized and delipidated BSA showed also no effect, crude commercial BSA preparation (Cohn fraction V) exhibited an ACTH-like cofactor effect which resulted in a marked increase in the net cortisol production by stimulated cells. These observations might be explained by the presence in crude BSA of lipoprotein-cholesterol complexes, possibly acting as an extracellular source of cholesterol available for corticosteroidogenesis. It may be concluded that specific high affinity cortisol binding systems present outside adrenocortical steroidogenic cells do not influence their secretory activity under short term in vitro condition. In addition, it can be stressed that use of ill defined protein preparations (e.g. crude BSA) may lead to artifactual observations in the study of the differentiated functions of isolated steroidogenic cells. PMID:6287106

  2. Evaluation of the radiosensitivity of acute myeloblastic leukaemia progenitor cells by culture methods exploring self-renewal. Evaluation de la radiosensibilite des progeniteurs de leucemie aigue myeloblastique par des methodes de culture explorant ou non l'autorenouvellement

    Energy Technology Data Exchange (ETDEWEB)

    Cowen, D.; Richaud, P.; Landriau, S.; Lagarde, P.; Gualde, N. (Fondation Bergonie, 33 - Bordeaux (France)); Boiron, J.M. (Hopital du Haut-Leveque, 33 - Pessac (France)); Mahon, F.X.; Belloc, F. (Hopital Regional, 33 - Bordeaux (France)); Reiffers, J. (Hopital du Haut-Leveque, 33 - Pessac (France) Hopital Regional, 33 - Bordeaux (France))

    1993-01-01

    The progenitor cells of acute myeloblastic leukaemia (AML) are usually cultured in methylcellulose which selects for terminal dividing cells. Suspension cultures have been developed because they reflect self-renewal: the exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. We have compared the radiosensitivity of the progenitors of AML grown either in methylcellulose alone or first in suspension for 7 days before being plated in methylcellulose. Cells were harvested from leukaemic bone marrows at the moment of diagnosis. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical and immunophenotypic analysis and by the use of growth factors which do not stimulate T-lymphocytes. The cell-cycle distribution of leukaemic blasts was comparable for all the samples. This method enabled aggressive leukaemias to be selected. The radiosensitivity showed wide variations from one patient to another (Do ranging from 0.35 to 2.6 Gy) whichever culture method used. The progenitor cells capable of self-renewal were more radiosensitive (Mean Do 0.9[+-]0.4 Gy) than terminal dividing cells (Mean Do = 1.35[+-]0.5 Gy). In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. The shape of the curves was better fitted by the linear quadratic model with very low values of [alpha]/[beta], suggesting a reduced antileukaemic effect in case of fractionation.

  3. Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

    Science.gov (United States)

    Zhao, Xiaozhi; Chen, Wei; Ge, Liyuan; Jiang, Wei; Tang, Bo; Zhang, Qing; Xu, Xiaoyu; Wang, Chong; Cao, Lin; Guo, Hongqian

    2016-08-01

    Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers. PMID:27113713

  4. A high affinity binding site for cytokinin to a particulate fraction in carrot suspension cells

    International Nuclear Information System (INIS)

    Carrot suspension cells contain one class of high affinity binding sites for cytokinin in an 80,000 X g particulate fraction. Binding of [8-14C] - benzylaminopurine (BA) to this fraction assayed by a sedimentation method was found to be optimal at ph 6.0 and thermolabile. Specific binding was proved in competition experiments in which labelled BA was displaced by increasing concentrations of unlabelled BA. Scatchard plots of these results displayed a dissociation constant (Ksub(d)) of 33+- 6 n.M. The number of binding sites found was 1,100+-120 fmol g-1 fresh weight which is equivalent to a frequency of 23,000 binding sites per cell. The specificity of the binding sites to cytokinins and their analogues followed the sequence BA with highest affinity, kinetin, zeatin, iP and adenine. The cytokinin ribosides generally had a lower affinity than their cytokinin bases, and the affinity decreased in the order [9 R] BA, [9 R] iP, [i R]Z, [9 R] A. (author)

  5. Cellular heredity in haploid cultures of somatic cells. Progress report, August 1978-September 1979

    International Nuclear Information System (INIS)

    During the past year we have continued our studies of the relation of ultraviolet mutagenesis to DNA repair in cultures of the haploid frog cell line ICR 2A. Our 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

  6. Cellular heredity in haploid cultures of somatic cells. Progress report, August 1978-September 1979

    International Nuclear Information System (INIS)

    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. Role of cellular antioxidants (glutathione and ascorbic acid) in the growth and development of wild carrot suspension cultures

    International Nuclear Information System (INIS)

    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 14C-2, 4-D. The embryo fraction had a lower concentration of 14C 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

  8. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    Directory of Open Access Journals (Sweden)

    Parvaneh Farzaneh

    2012-01-01

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

  9. Estimating the number of viable animal cells in multi-well cultures based on their lactate dehydrogenase activities.

    Science.gov (United States)

    Haslam, G; Wyatt, D; Kitos, P A

    2000-01-01

    A method is described for estimating the numbers ofanimal cells in multi-well culture by simultaneouslymeasuring the lactate dehydrogenase activity of thetotal culture and the medium. The difference betweenthe two reflects the dehydrogenase content of thecells and correlates with cell number. This LDH/INTmethod was tested using several lines of normal andtransformed suspension and adherent cells. Thelactate dehydrogenase activities of duplicate cultureswere determined colourimetrically using reactioncocktails containing lactate, NAD(+), diaphorase,and p-iodonitrotetrazolium violet, with and withoutTriton X-100. The difference in absorbance at 490 nm(DeltaA(490) = A(490, test) - A(490, control)) was used to calculate the lactatedehydrogenase activity of the total culture (+ Triton)and the medium (- Triton). The cellular lactatedehydrogenase activity (difference between totaland medium dehydrogenaseactivities) was proportional to viable cell number. The effects on cell growth of four metabolicinhibitors, sodium azide, actinomycin D,cycloheximide, and taxol, were determined using theLDH/INT assay and direct cell counting. The inhibitorconcentrations that caused decreases in the LDHactivity and cell number by 50% were similar. TheLDH/INT assay is quick and sensitive, works equallywell for adherent and suspension cells, and providesinformation about LDH activities of both the mediumand cells. It is particularly useful for screeningpotential cell-growth inhibitors. PMID:19002967

  10. Effect of Agrobacterium rhizogenes and elicitation on the asiaticoside production in cell cultures of Centella asiatica

    Directory of Open Access Journals (Sweden)

    Komar Ruslan

    2012-01-01

    Full Text Available 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 Cu 2+ 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 ΅M in cell suspension cultures at day 14 was up to 171% compared to explant and 494% compared to control callus; copper ion (25 ΅M 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.

  11. 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........

  12. 昆虫细胞(Sf21)悬浮培养过程中生长限制性基质 间歇补加技术的应用%Application of Intermittent-feeding of Growth-limiting Nutrients in Suspension Culture of Insect Cells(Sf21)

    Institute of Scientific and Technical Information of China (English)

    赵佼; 谭文松; 周燕; 周利; 俞俊棠

    2000-01-01

    On the basis of the growth and metabolism behavior inherent in suspended Sf21 insect cells,the intermittent feeding of growth-limiting nutrients(glucose and protein hydrolysates)was employed in the regulation of cellular growth during the cultivation by using the residual glucose concentration as a reference point. It was shown that as compared with the batch cultivation, the intermittent-feeding of growth-limiting nutrients effectively prolonged the growth and stationary phase for Sf21 insect cells grown in two representative culture medium(TC-100 and IPL-41). The maximum cell density was increased from 3.0×106 cells/ml to 6.5×106 cells/mL in TC-100 medium, and in IPL-41 medium, the maximum cell density was increased from 7.05×106 cells/mL to 9.0×106 cells/mL. As the defined nutrient solution was used for feeding in lieu of the complicated and expensive base medium, the technique would find prospects in large scale high-density cultivation of insect cells.%基于Sf21昆虫细胞在悬浮培养过程中所表现出的生长代谢特征,提出以培养液中残糖浓度作为控制参数,并利用限制性基质(葡萄糖和蛋白水解物)的间歇补加技术调控细胞生长的方案。实际控制表明:与批培养相比,Sf21细胞在两种具代表性的昆虫细胞培养基(IPL-41和TC-100)中的生长期和稳定期都得到了有效的延长。TC-100培养液中最高细胞培养密度由3.0×106 cells/mL提高到6.5×106 cells/mL;IPL-41培养液中最高细胞培养密度则由7.05×106 cells/mL提高到9.0×106cells/mL。由于限制性基质的间歇补加技术是利用较确定的营养成分来代替复杂昂贵的补料培养基,因此更适合于昆虫细胞的大规模高密度培养。

  13. Darwinian Evolution of Prions in Cell Culture*

    OpenAIRE

    Li, Jiali; Browning, Shawn; Mahal, Sukhvir P.; Oelschlegel, Anja M.; Weissmann, Charles

    2009-01-01

    Prions are infectious proteins consisting mainly of PrPSc, a β sheet-rich conformer of the normal host protein PrPC, and occur in different strains. Strain identity is thought to be encoded by PrPSc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating “mutants”, and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, ...

  14. Production of Phenolic Compounds in Cell Suspensions of Arctic Bramble and Cloudberry

    OpenAIRE

    Viitanen, Arto

    2015-01-01

    Phenolic compounds are of interest for various industries, and can be found in differing qualities and quantities between plant species, with particularly high concentrations observed in Nordic berries. The natural yields of these berries are highly vulnerable to environmental conditions, and consequently methods for producing these valuable compounds in bioreactors are being investigated. However, the phenolic content in all cultures is highly dependent on growth conditions and the used cell...

  15. Accumulation of Triterpenoids in Betula platyphylla Suspension Culture Induced by a Fungal Elicitor%真菌诱导子促进白桦悬浮细胞三萜的积累

    Institute of Scientific and Technical Information of China (English)

    翟俏丽; 范桂枝; 詹亚光

    2011-01-01

    Endophytic fungus of Phomopsis isolated from birch(Betula platyphylla) bark can be used as an elicitor to induce accumulation of triterpenoids in birch cell suspension culture. The change of dry weight and triterpenoids content of birch suspension cell were investigated at early growth phase, exponential growth phase and late growth phase after a fungal elicitor of 40, 100, and 400 (xg-mL"' concentration was added to birch suspension cells. Results showed that different induction schemes all promoted accumulation of triterpenoids, while the treatments reduced the dry weight of birch cells. Suspension cells at exponential growth phase were most easy to be induced, and 40 ng-mL"' fungal elicitor enhanced the triterpenoids content to 29. 47 mg-g"1, increasing by 789$j than that of the control. We investigated changes in pH and conductivity of the medium, and the activity of phenylalanine ammonialyase ( PAL) and peroxidase ( POD) under the optimal induction condition. Results showed that the fungal elicitor induced alkalinization of the medium and increased the conductivity, and significantly increased the activity of PAL and POD of birch suspension cells, which was 5. 70 and S. 74 fold as high as the control after 6 - 10 h of treatment, respectively. These results suggested that fungal elicitation induced defense reaction in birch suspension cell. General phenylpropanoid pathway and oxidative burst might involved in the triterpenoids accumulation induced by the fungal elicitor.%将促进白桦三萜积累的拟茎点霉属的内生真菌诱导子添加到白桦悬浮培养体系中,研究40,100,400pg·mL-1的真菌诱导子对生长初期、指数生长期和生长末期的悬浮细胞干质量和三萜积累的影响.结果表明:真菌诱导子的不同诱导方案均促进白桦悬浮细胞中三萜的积累,而细胞干质量积累却被抑制;其中最佳诱导条件为在指数生长期的白桦细胞中添加40 μg·mL-1的真菌诱导子诱导1天,诱导

  16. Fabrication and electrochemical performance of solid oxide fuel cell components by atmospheric and suspension plasma spray

    Institute of Scientific and Technical Information of China (English)

    XIA Wei-sheng; YANG Yun-zhen; ZHANG Hai-ou; WANG Gui-lan

    2009-01-01

    The theory of functionally graded material (FGM) was applied in the fabrication process of PEN (Positive- Electrolyte-Negative),the core component of solid oxide fuel cell (SOFC).To enhance its electrochemical performance,the functionally graded PEN of planar SOFC was prepared by atmospheric plasma spray (APS).The cross-sectional SEM micrograph and element energy spectrum of the resultant PEN were analyzed.Its interface resistance was also compared with that without the graded layers to investigate the electrochemical performance enhanced by the functionally graded layers.Moreover,a new process,suspension plasma spray (SPS) was applied to preparing the SOFC electrolyte.Higher densification of the coating by SPS,1.61%,is observed,which is helpful to effectively improve its electrical conductivity.The grain size of the electrolyte coating fabricated by SPS is also smaller than that by APS,which is more favourable to obtain the dense electrolyte coatings.To sum up,all mentioned above can prove that the hybrid process of APS and SPS could be a better approach to fabricate the PEN of SOFC stacks,in which APS is for porous electrodes and SPS for dense electrolyte.

  17. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    OpenAIRE

    Parvaneh Farzaneh; Laleh Nikfarjam

    2011-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma conta...

  18. A Combination of Culture Conditions and Gene Expression Analysis Can Be Used to Investigate and Predict hES Cell Differentiation Potential towards Male Gonadal Cells.

    Directory of Open Access Journals (Sweden)

    Kristín Rós Kjartansdóttir

    Full Text Available Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

  19. Surface Acoustic Waves (SAW-Based Biosensing for Quantification of Cell Growth in 2D and 3D Cultures

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2015-12-01

    Full Text Available Detection and quantification of cell viability and growth in two-dimensional (2D and three-dimensional (3D cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in

  20. 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.

  1. Stages of Cell Cannibalism--Entosis--in Normal Human Keratinocyte Culture.

    Science.gov (United States)

    Garanina, A S; Khashba, L A; Onishchenko, G E

    2015-11-01

    Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such "cell-in-cell" structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death. PMID:26615438

  2. Elicitors' influenced differential ginsenoside production and exudation into medium with concurrent Rg3/Rh2 panaxadiol induction in Panax quinquefolius cell suspensions.

    Science.gov (United States)

    Biswas, Tanya; Kalra, Alok; Mathur, A K; Lal, R K; Singh, Manju; Mathur, Archana

    2016-06-01

    Cobalt nitrate, nickel sulphate, hydrogen peroxide, sodium nitroprusside, and culture filtrates of Pseudomonas monteili, Bacillus circularans, Trichoderma atroviridae, and Trichoderma harzianum were tested to elicit ginsenoside production in a cell suspension line of Panax quinquefolius. Abiotic elicitors preferentially increased panaxadiols whereas biotic elicitors upregulated the panaxatriol synthesis. Cobalt nitrate (50 μM) increased total ginsenosides content by twofold (54.3 mg/L) within 5 days. It also induced the Rc synthesis that was absent in the control cultures. Elicitation with P. monteili (2.5 % v/v, 5 days) also supported 2.4-fold enhancement in saponin yield. Elicitation by T. atroviridae or hydrogen peroxide induced the synthesis of Rg3 and Rh2 that are absent in ginseng roots. The highest ginsenosides productivity (3.2-fold of control) was noticed in cells exposed to 1.25 % v/v dose of T. atroviridae for 5 days. Treating cells with T. harzianum for 15 days afforded maximum synthesis and leaching (8.1 mg/L) of ginsenoside Rh1. PMID:26795963

  3. Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96-well plates by facilitating oxygen transfer.

    Science.gov (United States)

    Meyer, Aaron; Condon, Russell G G; Keil, Gregory; Jhaveri, Nikita; Liu, Zhong; Tsao, Yung-Shyeng

    2012-01-01

    In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation. PMID:21954223

  4. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation

    Directory of Open Access Journals (Sweden)

    OLESIA O. GRYGORIEVA

    2015-05-01

    Full Text Available Abstract. Grygorieva OO, Berezovsjka MA, Dacenko OI. 2015. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation. Nusantara Bioscience 7: 38-42. Two cultures of Chlamydomonas actinochloris Deason et Bold in the lag-phase were exposed to the microwave irradiation. One of them (culture 1 was not treated beforehand, whereas the other (culture 2 was irradiated by microwaves 2 years earlier. The measurement of cell quantity as well as measurement of change of intensities and spectra of cultures photoluminescence (PL in the range of chlorophyll a emission was regularly conducted during the cell cultures development. Cell concentration of culture 1 exposed to the microwave irradiation for the first time has quickly restored while cell concentration of culture 2 which was irradiated repeatedly has fallen significantly. The following increasing of cell concentration of culture 2 is negligible. Cell concentration reaches the steady-state level that is about a half of the cell concentration of control culture. Initially the PL efficiency of cells of both cultures decreases noticeable as a result of irradiation. Then there is the monotonic increase to the values which are significantly higher than the corresponding values in the control cultures. The ratio of the intensities at the maxima of the main emission bands of chlorophyll for control samples of both cultures remained approximately at the same level. At the same time effect of irradiation on the cell PL spectrum appears as a temporary reduction of this magnitude.

  5. One-Step Detection of Major Lipid Components in Submicroliter Volumes of Unpurified Liposome and Cell Suspensions.

    Science.gov (United States)