WorldWideScience

Sample records for cell culture medium

  1. Characterization of conditioned medium of cultured bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Norihiko; Nakai, Yoshiyasu; Seo, Tae-Boem; Yamada, Yoshihiro; Ohno, Takayuki; Yamanaka, Atsuo; Nagai, Yoji; Fukushima, Masanori; Suzuki, Yoshiyuki; Nakatani, Toshio; Ide, Chizuka

    2010-10-08

    It has been recognized that bone marrow stromal cell (BMSC) transplantation has beneficial effects on spinal cord injury in animal models and therapeutic trials. It is hypothesized that BMSCs provide microenvironments suitable for axonal regeneration and secrete some trophic factors to rescue affected cells from degeneration. However, the molecular and cellular mechanisms of the trophic factors involved remain unclear. In the present study, we examined the effects of trophic factors secreted by rat BMSCs using bioassays involving cultured hippocampal neurons. The conditioned medium (CM) as well as non-contact co-culture of BMSCs promoted neurite outgrowth and suppressed TUNEL-positive cells compared to serum-free D-MEM. Protein analyses of the CM by antibody-based protein array analysis and ELISA revealed that the CM contained insulin-like growth factor (IGF)-1, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-beta1. DNA microarray analysis revealed that neurons highly expressed receptors of IGF-1 and TGF-beta1. However, their expression indices remained unchanged even after the CM treatment. The individual trophic factors mentioned above or their combinations were less effective at promoting neuronal survival and neurite outgrowth than the CM. The present study showed that BMSCs secreted various kinds of molecules into the culture medium including trophic factors to promote neuronal survival and neurite outgrowth. The main trophic factors responsible remain to be elucidated. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  2. Protective layer formation on magnesium in cell culture medium

    Energy Technology Data Exchange (ETDEWEB)

    Wagener, V.; Virtanen, S., E-mail: virtanen@ww.uni-erlangen.de

    2016-06-01

    In the past, different studies showed that hydroxyapatite (HA) or similar calcium phosphates can be precipitated on Mg during immersion in simulated body fluids. However, at the same time, in most cases a dark grey or black layer is built under the white HA crystals. This layer seems to consist as well of calcium phosphates. Until now, neither the morphology nor its influence on Mg corrosion have been investigated in detail. In this work commercially pure magnesium (cp) was immersed in cell culture medium for one, three and five days at room temperature and in the incubator (37 °C, 5% CO{sub 2}). In addition, the influence of proteins on the formation of a corrosion layer was investigated by adding 20% of fetal calf serum (FCS) to the cell culture medium in the incubator. In order to analyze the formed layers, SEM images of cross sections, X-ray Photoelectron Spectroscopy (XPS), X-ray diffraction (XRD), Energy Dispersive X-ray Spectroscopy (EDX) and Fourier Transformed Infrared Spectroscopy (FTIR) measurements were carried out. Characterization of the corrosion behavior was achieved by electrochemical impedance spectroscopy (EIS) and by potentio-dynamic polarization in Dulbecco's Modified Eagle's Medium (DMEM) at 37 °C. Surface analysis showed that all formed layers consist mainly of amorphous calcium phosphate compounds. For the immersion at room temperature the Ca/P ratio indicates the formation of HA, while in the incubator probably pre-stages to HA are formed. The different immersion conditions lead to a variation in layer thicknesses. However, electrochemical characterization shows that the layer thickness does not influence the corrosion resistance of magnesium. The main influencing factor for the corrosion behavior is the layer morphology. Thus, immersion at room temperature leads to the highest corrosion protection due to the formation of a compact outer layer. Layers formed in the incubator show much worse performances due to completely porous

  3. The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan; Doeselaar, Marina van; Meij, Björn; Tryfonidou, M; Ito, Keita

    2015-01-01

    OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  4. The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, K

    2016-01-01

    Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  5. Tryptophan oxidation catabolite, N-formylkynurenine, in photo degraded cell culture medium results in reduced cell culture performance.

    Science.gov (United States)

    McElearney, Kyle; Ali, Amr; Gilbert, Alan; Kshirsagar, Rashmi; Zang, Li

    2016-01-01

    Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers.

  6. Dissolved oxygen concentration in the medium during cell culture: Defects and improvements.

    Science.gov (United States)

    Zhang, Kuan; Zhao, Tong; Huang, Xin; He, Yunlin; Zhou, Yanzhao; Wu, Liying; Wu, Kuiwu; Fan, Ming; Zhu, Lingling

    2016-03-01

    In vitro cell culture has provided a useful model to study the effects of oxygen on cellular behavior. However, it remains unknown whether the in vitro operations themselves affect the medium oxygen levels and the living states of cells. In addition, a prevailing controversy is whether reactive oxygen species (ROS) production is induced by continuous hypoxia or reoxygenation. In this study, we have measured the effects of different types of cell culture containers and the oxygen environment where medium replacement takes place on the actual oxygen tension in the medium. We found that the deviations of oxygen concentrations in the medium are much greater in 25-cm(2) flasks than in 24-well plates and 35-mm dishes. The dissolved oxygen concentrations in the medium were increased after medium replacement in normoxia, but remained unchanged in glove boxes in which the oxygen tension remained at a low level (11.4, 5.7, and 0.5% O2 ). We also found that medium replacement in normoxia increased the number of ROS-positive cells and reduced the cell viability; meanwhile, medium replacement in a glove box did not produce the above effects. Therefore, we conclude that the use of 25-cm(2) flasks should be avoided and demonstrate that continuous hypoxia does not produce ROS, whereas the reoxygenation that occurs during the harvesting of cells leads to ROS and induces cell death. © 2015 International Federation for Cell Biology.

  7. Aquatic flower-inspired cell culture platform with simplified medium exchange process for facilitating cell-surface interaction studies.

    Science.gov (United States)

    Hong, Hyeonjun; Park, Sung Jea; Han, Seon Jin; Lim, Jiwon; Kim, Dong Sung

    2016-02-01

    Establishing fundamentals for regulating cell behavior with engineered physical environments, such as topography and stiffness, requires a large number of cell culture experiments. However, cell culture experiments in cell-surface interaction studies are generally labor-intensive and time-consuming due to many experimental tasks, such as multiple fabrication processes in sample preparation and repetitive medium exchange in cell culture. In this work, a novel aquatic flower-inspired cell culture platform (AFIP) is presented. AFIP aims to facilitate the experiments on the cell-surface interaction studies, especially the medium exchange process. AFIP was devised to capture and dispense cell culture medium based on interactions between an elastic polymer substrate and a liquid medium. Thus, the medium exchange can be performed easily and without the need of other instruments, such as a vacuum suction and pipette. An appropriate design window of AFIP, based on scaling analysis, was identified to provide a criterion for achieving stability in medium exchange as well as various surface characteristics of the petal substrates. The developed AFIP, with physically engineered petal substrates, was also verified to exchange medium reliably and repeatedly. A closed structure capturing the medium was sustained stably during cell culture experiments. NIH3T3 proliferation results also demonstrated that AFIP can be applied to the cell-surface interaction studies as an alternative to the conventional method.

  8. Freeform micropatterning of living cells into cell culture medium using direct inkjet printing.

    Science.gov (United States)

    Park, Ju An; Yoon, Sejeong; Kwon, Jimin; Now, Hesung; Kim, Young Kwon; Kim, Woo-Jong; Yoo, Joo-Yeon; Jung, Sungjune

    2017-11-06

    Microfabrication methods have widely been used to control the local cellular environment on a micron scale. However, accurately mimicking the complexity of the in vivo tissue architecture while maintaining the freedom of form and design is still a challenge when co-culturing multiple types of cells on the same substrate. For the first time, we present a drop-on-demand inkjet printing method to directly pattern living cells into a cell-friendly liquid environment. High-resolution control of cell location is achieved by precisely optimizing printing parameters with high-speed imaging of cell jetting and impacting behaviors. We demonstrated the capabilities of the direct cell printing method by co-printing different cells into various designs, including complex gradient arrangements. Finally, we applied this technique to investigate the influence of the heterogeneity and geometry of the cell population on the infectivity of seasonal H1N1 influenza virus (PR8) by generating A549 and HeLa cells printed in checkboard patterns of different sizes in a medium-filled culture dish. Direct inkjet cell patterning can be a powerful and versatile tool for both fundamental biology and applied biotechnology.

  9. Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium

    NARCIS (Netherlands)

    Egorova-Zachernyuk, T.A.; Bosman, G.J.C.G.M.; Grip, W.J. de

    2011-01-01

    Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids

  10. Cell culture medium as an alternative to conventional simulated body fluid.

    Science.gov (United States)

    Lee, Juliana T Y; Leng, Yang; Chow, King L; Ren, Fuzeng; Ge, Xiang; Wang, Kefeng; Lu, Xiong

    2011-06-01

    Simulated body fluid (SBF) has been widely used for bioactivity assessment of biomaterials. Many different recipes have been developed, but there is still room for improvement. We have suggested the use of cell culture medium to overcome the drawbacks of conventional SBF. Compared with conventional SBF, cell culture medium is easy to prepare. The normal practices in cell culturing, such as filtering, can eliminate insoluble precipitates in the medium and incubation at 37 °C in an atmosphere with 5% CO(2) also better simulates the in vivo environment. After 4 days immersion in carbonate buffered Dulbecco's modified Eagle's medium (DMEM), precipitates were found to have formed on the surfaces of hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). In order to further verify the use of cell culture medium for SBF studies, we carried out computational thermodynamic and kinetic analyses of the precipitation reaction to reveal the effect of pH and ion concentrations on the driving force and nucleation rate of precipitation of different calcium phosphates (CaP). In general, a slight increase in pH of the cell culture medium from physiological pH (pH 7.4) would favor CaP precipitation thermodynamically and increase the rate, as in the case of r-SBF reported previously. [Ca] and [P] have more impact on precipitation compared with other ions, but the effect is consistent among different materials, indicating that other cell culture media with slightly different compositions may also be used. This study also shows that matching the buffer with the environment is required and fetal bovine serum (FBS) slows down surface CaP precipitation on HA. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  11. Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions.

    Science.gov (United States)

    Bovo, R; Galesi, A L L; Jorge, S A C; Piccoli, R A M; Moraes, A M; Pereira, C A; Augusto, E F P

    2008-05-01

    In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II((R)) and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the calculation of kinetic parameters. The results show that S2 cells metabolism was able to adjust to different environmental situations, as determined by medium formulation, as well as by the particular situation resulting from the culture conditions. Cells attained a 163% higher final cell concentration (1.4 x 10(7) cells mL(-1)) in SF900 II((R)) medium, when compared to serum-supplemented TC-100 medium. Also, a maximum specific cell growth rate 52% higher in SF900 II((R) )medium, when compared to serum-supplemented TC-100 one, was observed. Glutamine was the growth limiting factor in SF900 II((R)) medium, while glucose, sometimes associated with glutamine, controlled growth in serum-supplemented TC-100 medium based formulation. The different pattern of lactate production is an example of the versatility of the metabolism of these cells. This by-product was produced only in glutamine limitation, but the amount synthesized depended not only on the excess glucose, but on other medium components. Therefore, in serum-supplemented TC-100 medium a much smaller lactate amount was generated. Besides, glucose was identified not only as a growth limiting factor, but also as a viability limiting factor, since its depletion accelerated cell death.

  12. Enhanced growth medium and method for culturing human mammary epithelial cells

    Science.gov (United States)

    Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  13. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    Science.gov (United States)

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference.

  14. Microfluidic cell culture chip with multiplexed medium delivery and efficient cell/scaffold loading mechanisms for high-throughput perfusion 3-dimensional cell culture-based assays.

    Science.gov (United States)

    Huang, Song-Bin; Wu, Min-Hsien; Wang, Shih-Siou; Lee, Gwo-Bin

    2011-06-01

    This study reports a microfluidic cell culture chip consisting of 48 microbioreactors for high-throughput perfusion 3-dimensional (3-D) cell culture-based assays. Its advantages include the capability for multiplexed and backflow-free medium delivery, and both efficient and high-throughput micro-scale, 3-D cell culture construct loading. In this work, the microfluidic cell culture chip is fabricated using two major processes, specifically, a computer-numerical-controlled (CNC) mold machining process and a polydimethylsiloxane (PDMS) replication process. The chip is composed of micropumps, microbioreactors, connecting microchannels and a cell/agarose scaffold loading mechanism. The performance of the new pneumatic micropumps and the cell/agarose scaffold loading mechanism has been experimentally evaluated. The experimental results show that this proposed multiplexed medium-pumping design is able to provide a uniform pumping rate ranging from 1.5 to 298.3 μl hr(-1) without any fluid backflow and the resultant medium contamination. In addition, the simple cell/agarose loading method has been proven to be able to load the 3-D cell culture construct uniformly and efficiently in all 48 microbioreactors investigated. Furthermore, a micro-scale, perfusion, 3-D cell culture-based assay has been successfully demonstrated using this proposed cell culture chip. The experimental results are also compared to a similar evaluation using a conventional static 3-D cell culture with a larger scale culture. It is concluded that the choice of a cell culture format can influence assay results. As a whole, because of the inherent advantages of a miniaturized perfusion 3-D cell culture assay, the cell culture chip not only can provide a stable, well-defined and more biologically-meaningful culture environment, but it also features a low consumption of research resources. Moreover, due to the integrated medium pumping mechanism and the simple cell/agarose loading method, this chip is

  15. Plasma generated in culture medium induces damages of HeLa cells due to flow phenomena

    Science.gov (United States)

    Sato, Yusuke; Sato, Takehiko; Yoshino, Daisuke

    2018-03-01

    Plasma in a liquid has been anticipated as an effective tool for medical applications, however, few reports have described cellular responses to plasma generated in a liquid similar to biological fluids. Herein we report the effects of plasma generated in a culture medium on HeLa cells. The plasma in the culture medium produced not only heat, shock waves, and reactive chemical species but also a jet flow with sub millimeter-sized bubbles. Cells exposed to the plasma exhibited detachment, morphological changes, and changes in the actin cytoskeletal structure. The experimental results suggest that wall shear stress over 160 Pa was generated on the surface of the cells by the plasma. It is one of the main factors that cause those cellular responses. We believe that our findings would provide valuable insight into advancements in medical applications of plasma in a liquid.

  16. Differentiation behavior of iPS cells cultured on PLGA with osteoinduction medium.

    Science.gov (United States)

    Tokita, Reiko; Nakajima, Kei; Inoue, Kenji; Al-Wahabi, Akram; Ser-Od, Tungalag; Matsuzaka, Kenichi; Inoue, Takashi

    2017-01-31

    In the present report, we have generated osteoblast-like cells derived from mouse induced-pluripotent stem (iPS) cells on PLGA with osteoinduction medium in vitro and in vivo. The cell culture period was 2 weeks. At 2 weeks, mRNA level of type I collagen was significantly higher than at 1 week. Osteocalcin mRNA level at 2 weeks was tendency to increase compared with at 1 week. And the cells cultured on PLGA were positive for immunofluorescent staining of osteocalcin and alizarin red S staining. The scaffold and osteogenic-like cells induced in vitro were implanted subcutaneously into SCID mice. In resected teratoma, hard tissues resembling bone were observed mixed with other tissues on the scaffold. The sum of these findings suggests that PLGA does not disturb the osteogenesis of iPS cells.

  17. Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium

    OpenAIRE

    Kendall, Michaela; Hodges, Nikolas J.; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

    2015-01-01

    When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of ...

  18. Growth factor-defined culture medium for human mesenchymal stem cells.

    Science.gov (United States)

    Mimura, Sumiyo; Kimura, Naohiro; Hirata, Mitsuhi; Tateyama, Daiki; Hayashida, Midori; Umezawa, Akihiro; Kohara, Arihiro; Nikawa, Hiroki; Okamoto, Tetsuji; Furue, Miho K

    2011-01-01

    Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.

  19. Human dental pulp stem cells cultured in serum-free supplemented medium

    Directory of Open Access Journals (Sweden)

    Virginie eBonnamain

    2013-12-01

    Full Text Available Growing evidence show that human dental pulp stem cells (DPSCs could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 hours. Adherent (ADH and non-adherent (non-ADH cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF and basic fibroblast growth factor (bFGF. Both ADH and non-ADH populations were analyzed by FACS and/or PCR.Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133 and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expended and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte precursors at different stages of commitment and interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the

  20. Prolactin inhibits the steroidogenesis in midfollicular phase human granulosa cells cultured in a chemically defined medium.

    Science.gov (United States)

    Cutie, E; Andino, N A

    1988-04-01

    In vitro studies were conducted on prolactin (PRL) effects on human granulosa cell steroidogenesis. Cells derived from healthy midfollicular phase follicles were cultured in a chemically defined medium supplemented with androstenedione (delta 4 A) 10(-7) M. Cultures treated with follicle-stimulating hormone (FSH) showed a dose-dependent increase of estradiol (E2) and progesterone (P) secretion. The authors demonstrated that PRL (greater than or equal to 10 ng/ml) inhibits basal as well as FSH (10 ng/ml)-stimulated E2 and P secretion. This PRL effect was overcome only by FSH maximal stimulating doses (100 ng/ml). These results suggest a direct inhibitory effect of PRL on granulosa cell steroidogenesis acting as a negative modulator of FSH action. These effects might be related to the ovarian dysfunction observed in hyperprolactinemia.

  1. The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture.

    Science.gov (United States)

    de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, Keita

    2016-01-01

    Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their native environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect on extracellular matrix production. Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 μg/mL Link N. Furthermore, BM and NCCM samples were injected with 10(6) BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, glycosaminoglycan [GAG], hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC-injected groups was not higher than in the other groups after 4 weeks of culture. This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration.

  2. Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells.

    Science.gov (United States)

    Tokuyasu, K; Kaneko, S; Hayashi, K; Mori, Y

    1999-09-10

    With the aid of a signal sequence of a chitinase from Streptomyces lividans, a recombinant chitin deacetylase, whose gene originated from a Deuteromycete, Colletotrichum lindemuthianum, was produced in the culture medium of Escherichia coli cells, existing as a highly active form without the signal peptide. During the production of the recombinant chitin deacetylase, both a slight increase in the value of OD600 nm in the culture medium and a drastic decrease in viable cell number were observed. When penta-N-acetyl-chitopentaose was used as the substrate, the recombinant chitin deacetylase had comparable kinetic parameters to those of the original enzyme from the fungus. The addition of a C-terminal six histidine sequence to the recombinant enzyme caused a slight decrease in the kcat value, and the further addition of a 12 amino acid sequence at its N-terminus caused a further decrease in the value. This production system allowed us to easily produce in the culture media the recombinant chitin deacetylases possessing as good properties as the original enzyme, without any disruption steps of the E. coli cells.

  3. Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

    International Nuclear Information System (INIS)

    Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

    2011-01-01

    The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25–200 μg/mL) and incubation time (0–72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

  4. Formation of industrial mixed culture biofilm in chlorophenol cultivated medium of microbial fuel cell

    Science.gov (United States)

    Hassan, Huzairy; Jin, Bo; Dai, Sheng; Ngau, Cornelius

    2016-11-01

    The formation of microbial biofilm while maintaining the electricity output is a challenging topic in microbial fuel cell (MFC) studies. This MFC critical factor becomes more significant when handling with industrial wastewater which normally contains refractory and toxic compounds. This study explores the formation of industrial mixed culture biofilm in chlorophenol cultivated medium through observing and characterizing microscopically its establishment on MFC anode surface. The mixed culture was found to develop its biofilm on the anode surface in the chlorophenol environment and established its maturity and dispersal stages with concurrent electricity generation and phenolic degradation. The mixed culture biofilm engaged the electron transfer roles in MFC by generating current density of 1.4 mA/m2 and removing 53 % of 2,4-dichlorophenol. The results support further research especially on hazardous wastewater treatment using a benign and sustainable method.

  5. LED-CT Scan for pH Distribution on a Cross-Section of Cell Culture Medium

    OpenAIRE

    Higashino, Nobuya; Takayama, Toshio; Ito, Hiroaki; Horade, Mitsuhiro; Yamaguchi, Yasutaka; Dylan Tsai, Chia-Hung; Kaneko, Makoto

    2018-01-01

    In cell culture, the pH of the culture medium is one of the most important conditions. However, the culture medium may have non-uniform pH distribution due to activities of cells and changes in the environment. Although it is possible to measure the pH distribution with an existing pH meter using distributed electrodes, the method involves direct contact with the medium and would greatly increase the risk of contamination. Here in this paper, we propose a computed tomography (CT) scan for mea...

  6. HepG2 cells develop signs of riboflavin deficiency within four days of culture in riboflavin-deficient medium*

    OpenAIRE

    Werner, Ricarda; Manthey, Karoline C.; Griffin, Jacob B.; Zempleni, Janos

    2005-01-01

    Flavin mononucleotide and flavin adenine dinucleotide are essential coenzymes in redox reactions. For example, flavin adenine dinucleotide is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells. Cells were cultured in riboflavin-deficient (3.1 nmol/L) medium for up to six days; controls ...

  7. Culture medium modulates the behaviour of human dental pulp-derived cells: Technical Note

    Directory of Open Access Journals (Sweden)

    S Lopez-Cazaux

    2006-02-01

    Full Text Available In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8mM Ca and 1mM Pi and RPMI 1640 (0.8mM Ca and 5mM Pi on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of -smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

  8. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    International Nuclear Information System (INIS)

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-01-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  9. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    Energy Technology Data Exchange (ETDEWEB)

    Aslanova, Afag [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Takagi, Ryo; Yamato, Masayuki; Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yamamoto, Masakazu, E-mail: yamamoto.ige@twmu.ac.jp [Department of Surgery, Institute of Gastroenterology, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  10. Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium.

    Science.gov (United States)

    Kendall, Michaela; Hodges, Nikolas J; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

    2015-02-05

    When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  11. Different arabinogalactan proteins (AGPs) are present in carrot (Daucus carota L.) cell culture medium and in seeds

    NARCIS (Netherlands)

    Immerzeel, P.; Schols, H.A.; Voragen, A.G.J.; Vries, de S.C.

    2004-01-01

    Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot (Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively

  12. Medium from X-rayed cultures induces DNA strand-breaks in non-irradiated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ikushima, T.; Okuyama, K.; Tanizaki, Y. [Kyoto Univ., Kyoto (Japan)

    2002-07-01

    There is growing evidence to indicate that several types of responses are induced by ionizing radiation in non-irradiated cells. Such bystander effects include the killing of non-irradiated cells, the induction of sister chromatid exchanges and chromosomal aberrations, and the induction of gene mutations and chromosomal instability and enhanced cell growth. In the present study, we assessed whether the medium from irradiated cultures can induce DNA strand-breaks in non-irradiated cells, using single-cell gel electrophoresis assay (comet assay). HeLa cells in culture were irradiated with 0.5 to 8 Gy of 140 kVp X-rays and one hour later, the medium was taken from the irradiated culture, passed through a filter and transferred to the parallel culture of non-irradiated HeLa cells as non-target cells. After incubation for 30 min, the comet assay was performed under alkaline and neutral conditions. Such treatments resulted in a dose-dependent increase in tail moment under either alkaline or neutral condition, indicating the induction of DNA single- or double-strand breaks, respectively. It was also shown that the clonogenic survival was reduced in the cells cultured in the medium from irradiated cultures. Such a change was not detected at all when medium alone was irradiated. These results provided disputed evidence that irradiated cells released certain genotoxic factor(s) into the culture medium that can induce DNA strand breaks leading to cell death. Our results suggest that physical contact between irradiated and non-irradiated cells may not be necessary for the bystander effects observed in this study. It appears that bystander responses may be mediated by multiple mechanisms.

  13. Medium from X-rayed cultures induces DNA strand-breaks in non-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Ikushima, T.; Okuyama, K.; Tanizaki, Y.

    2002-01-01

    There is growing evidence to indicate that several types of responses are induced by ionizing radiation in non-irradiated cells. Such bystander effects include the killing of non-irradiated cells, the induction of sister chromatid exchanges and chromosomal aberrations, and the induction of gene mutations and chromosomal instability and enhanced cell growth. In the present study, we assessed whether the medium from irradiated cultures can induce DNA strand-breaks in non-irradiated cells, using single-cell gel electrophoresis assay (comet assay). HeLa cells in culture were irradiated with 0.5 to 8 Gy of 140 kVp X-rays and one hour later, the medium was taken from the irradiated culture, passed through a filter and transferred to the parallel culture of non-irradiated HeLa cells as non-target cells. After incubation for 30 min, the comet assay was performed under alkaline and neutral conditions. Such treatments resulted in a dose-dependent increase in tail moment under either alkaline or neutral condition, indicating the induction of DNA single- or double-strand breaks, respectively. It was also shown that the clonogenic survival was reduced in the cells cultured in the medium from irradiated cultures. Such a change was not detected at all when medium alone was irradiated. These results provided disputed evidence that irradiated cells released certain genotoxic factor(s) into the culture medium that can induce DNA strand breaks leading to cell death. Our results suggest that physical contact between irradiated and non-irradiated cells may not be necessary for the bystander effects observed in this study. It appears that bystander responses may be mediated by multiple mechanisms

  14. Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium

    Energy Technology Data Exchange (ETDEWEB)

    Eberbeck, D; Zirpel, P; Trahms, L [Physikalisch-Technische Bundesanstalt, Abbestrasse 2-12, 10587 Berlin (Germany); Kettering, M; Hilger, I [Institute of Diagnostic and Interventional Radiology, University Hospital Jena, Erlanger Allee 101, 07747 Jena (Germany); Bergemann, C, E-mail: dietmar.eberbeck@ptb.d [Chemicell GmbH, Eresburgstrasse 22-23, 12103 Berlin (Germany)

    2010-10-13

    The knowledge of the physico-chemical characteristics of magnetic nanoparticles (MNPs) is essential to enhance the efficacy of MNP-based therapeutic treatments (e.g. magnetic heating, magnetic drug targeting). According to the literature, the MNP uptake by cells may depend on the coating of MNPs, the surrounding medium as well as on the aggregation behaviour of the MNPs. Therefore, in this study, the aggregation behaviour of MNPs in various media was investigated. MNPs with different coatings were suspended in cell culture medium (CCM) containing fetal calf serum (FCS) and the distribution of the hydrodynamic sizes was measured by magnetorelaxometry (MRX). FCS as well as bovine serum albumin (BSA) buffer (phosphate buffered saline with 0.1% bovine serum albumin) may induce MNP aggregation. Its strength depends crucially on the type of coating. The degree of aggregation in CCM depends on its FCS content showing a clear, local maximum at FCS concentrations, where the IgG concentration (part of FCS) is of the order of the MNP number concentration. Thus, we attribute the observed aggregation behaviour to the mechanism of agglutination of MNPs by serum compartments as for example IgG. No aggregation was induced for MNPs coated with dextran, polyarabic acid or sodium phosphate, respectively, which were colloidally stable in CCM.

  15. Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium

    International Nuclear Information System (INIS)

    Eberbeck, D; Zirpel, P; Trahms, L; Kettering, M; Hilger, I; Bergemann, C

    2010-01-01

    The knowledge of the physico-chemical characteristics of magnetic nanoparticles (MNPs) is essential to enhance the efficacy of MNP-based therapeutic treatments (e.g. magnetic heating, magnetic drug targeting). According to the literature, the MNP uptake by cells may depend on the coating of MNPs, the surrounding medium as well as on the aggregation behaviour of the MNPs. Therefore, in this study, the aggregation behaviour of MNPs in various media was investigated. MNPs with different coatings were suspended in cell culture medium (CCM) containing fetal calf serum (FCS) and the distribution of the hydrodynamic sizes was measured by magnetorelaxometry (MRX). FCS as well as bovine serum albumin (BSA) buffer (phosphate buffered saline with 0.1% bovine serum albumin) may induce MNP aggregation. Its strength depends crucially on the type of coating. The degree of aggregation in CCM depends on its FCS content showing a clear, local maximum at FCS concentrations, where the IgG concentration (part of FCS) is of the order of the MNP number concentration. Thus, we attribute the observed aggregation behaviour to the mechanism of agglutination of MNPs by serum compartments as for example IgG. No aggregation was induced for MNPs coated with dextran, polyarabic acid or sodium phosphate, respectively, which were colloidally stable in CCM.

  16. 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.

  17. The selective effect of plasma activated medium in an in vitro co-culture of liver cancer and normal cells

    Science.gov (United States)

    Duan, J.; Lu, X.; He, G.

    2017-01-01

    In this work, a co-culture system with liver cancer cell line HepG2 and normal cell line L02 is used to investigate the selective effect on cancer and normal cells by plasma activated medium (PAM), which is closer to the real environment where cancer cells develop. Besides, the co-culture system is a better model to study the selective effect than the widely used separate culture systems, where the cancer cell line and normal cell line are cultured independently. By using the co-culture system, it is found that there is an optimum dose of PAM to induce significant cancer cell apoptosis while keeping minimum damage to normal cells.

  18. Growth and maintenance of an embryogenic cell culture of daylily (Hemerocallis) on hormone-free medium

    Science.gov (United States)

    Smith, D. L.; Krikorian, A. D.

    1991-01-01

    Callus cultures of the diploid daylily (Hemerocallis) clone Autumn Blaze' were initiated and maintained in hormone-containing nutrient medium. At various times (from 6 weeks to 1 year) after being initiated, hormone-derived cultures were evaluated for their ability to be maintained and to multiply on hormone-free medium at low pH (between pH 4 and 4.5). Cultures had to be exposed to hormone-containing medium for at least 12 weeks before they could be maintained on hormone-free medium at low pH. The transition to maintainability on low pH hormone-free medium included the production of many aberrant embryonal forms ( neomorphs'). However, all hormone-derived cultures tested consisted entirely of preglobular stage proembryos (PGSPs) after 12-24 weeks on low pH hormone-free medium. PGSP cultures have been maintained and multiplied as such for over 1 year on low pH hormone-free medium. PGSPs continue their development into various somatic embryo stages when cultured on hormone-free medium buffered at pH 5.8. The production of well-formed somatic embryos was greatly enhanced when PGSPs were plated on activated charcoal impregnated filter papers that were placed on top of the agar surface. The gross morphology and histology of the PGSPs and stages of somatic embryo development are presented. The work shows that the ability of hormone-free medium at low pH to permit PGSP multiplication without development into later stages of embryo development is not restricted to carrot.

  19. Conditioned medium derived from mesenchymal stem cells culture as a intravesical therapy for cystitis interstitials.

    Science.gov (United States)

    Adamowicz, Jan; Pokrywczyńska, Marta; Drewa, Tomasz

    2014-06-01

    The treatment of Interstinal Cystitisis (IC) is still challenge for urologist. Available therapies do not result in long-term control of symptoms and do not provide pain relive to patients. Unique abilities of mesenchymal stem cells (MSC) could be used to develop new treatment approaches for Interstitial Cystitis. Conditioned Medium (CM) derived from MSC culture is rich in plenty of growth factors, cytokines and trophic agents which were widely reported to enhance regeneration of urinary bladder in different conditions. This ready mixture of growth factors could be used to develop intravesical therapy for patients with IC. MSC-CM has anti-apoptotic, anti-inflammatory, supportive, angiogenic, immunosuppressive and immunomodulative properties and seems to be ideal substance to prevent IC recurrence and to create favorable environment for regeneration of damaged bladder wall. Copyright © 2014. Published by Elsevier Ltd.

  20. Corrosion behavior of HPT-deformed TiNi alloys in cell culture medium

    Science.gov (United States)

    Shri, D. N. Awang; Tsuchiya, K.; Yamamoto, A.

    2017-09-01

    In recent years there are growing interest in fabrication of bulk nanostructured metals and alloys by using severe plastic deformation (SPD) techniques as new alternative in producing bulk nanocrystalline materials. These techniques allows for processing of bulk, fully dense workpiece with ultrafine grains. Metal undergoes SPD processing in certain techniques such as high pressure torsion (HPT), equal-channel angular pressing (ECAP) or multi-directional forging (MDF) are subjected to extensive hydrostatic pressure that may be used to impart a very high strain to the bulk solid without the introduction of any significant change in overall dimension of the sample. The change in the structure (small grain size and high-volume fraction of grain boundaries) of the material may result in the corrosion behavior different from that of the coarse-grained material. Electrochemical measurements were done to understand the corrosion behavior of TiNi alloys before and after HPT deformation. The experiment was carried out using standard three electrode setup (a sample as working electrode; a platinum wire as a counter electrode and a saturated calomel electrode in saturated KCl as a reference electrode) with the surface area of 26.42 mm2 exposed to the EMEM+10% FBS cell culture medium. The measurements were performed in an incubator with controlled environment at 37 °C and 5% CO2, simulating the cell culture condition. The potential of the specimen was monitored over 1 hour, and the stabilized potential was used as the open-circuit potential (EOCP). Potentiodynamic curves were scanned in the potential range from -0.5 V to 1.5 V relative to the EOCP, at a rate of 0.5 mV/s. The result of OCP-time measurement done in the cell culture medium shows that the OCP of HPT-deformed samples shifts towards to the more positive rather than that of BHPT samples. The OCP of deformed samples were ennobled to more than +70 mV for Ti-50mol%. The shift of OCP towards the nobler direction

  1. Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

    Science.gov (United States)

    Sands, Sandra S.; Meek, William D.; Hayashi, Jun; Ketchum, Robert J.

    2005-08-01

    Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4 5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 [mu]g/ml), transferrin (10 [mu]g/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

  2. AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM BY ION-EXCHANGE MEMBRANES

    Science.gov (United States)

    Metabolites such as ammonia and lactic acid formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. Cell culture conducted in the presence of such accumulated metabolites is therefore limited in pro...

  3. LED-CT Scan for pH Distribution on a Cross-Section of Cell Culture Medium.

    Science.gov (United States)

    Higashino, Nobuya; Takayama, Toshio; Ito, Hiroaki; Horade, Mitsuhiro; Yamaguchi, Yasutaka; Dylan Tsai, Chia-Hung; Kaneko, Makoto

    2018-01-11

    In cell culture, the pH of the culture medium is one of the most important conditions. However, the culture medium may have non-uniform pH distribution due to activities of cells and changes in the environment. Although it is possible to measure the pH distribution with an existing pH meter using distributed electrodes, the method involves direct contact with the medium and would greatly increase the risk of contamination. Here in this paper, we propose a computed tomography (CT) scan for measuring pH distribution using the color change of phenol red with a light-emitting diode (LED) light source. Using the principle of CT scan, we can measure pH distribution without contacting culture medium, and thus, decrease the risk of contamination. We have developed the device with a LED, an array of photo receivers and a rotation mechanism. The system is firstly calibrated with different shapes of wooden objects that do not pass light, we succeeded in obtaining their 3D topographies. The system was also used for measuring a culture medium with two different pH values, it was possible to obtain a pH distribution that clearly shows the boundary.

  4. Culture of transgenic Drosophila melanogaster Schneider 2 cells in serum-free media based on TC100 basal medium.

    Science.gov (United States)

    Galesi, Adriana L L; Pereira, Carlos A; Moraes, Angela M

    2007-11-01

    Requirements of eliminating animal proteins from cell culture have intensified in recent years, with the pressure of regulatory agencies related to biopharmaceuticals production. In this work, the substitution of fetal bovine serum by yeastolate and a soy hydrolysate (Hy Soy) for the culture of Drosophila melanogaster Schneider 2 cells transfected for the production of rabies virus G glycoprotein was evaluated. TC100 supplemented with glucose, glutamine, lipid emulsion and Pluronic F68 was employed as basal medium. Results show that yeastolate was more efficient on cell growth stimulation than Hy Soy. Cells adapted in medium formulation supplemented with 3 g/L yeastolate, 1% lipid emulsion, 10 g/L glucose, 3.5 g/L glutamine and 0.1% Pluronic F68 attained a maximum concentration of 10.7 x 10(6) cells/mL, with the expression of 9.4 ng/mL G glycoprotein.

  5. Prototypical antipsychotic drugs protect hippocampal neuronal cultures against cell death induced by growth medium deprivation

    Directory of Open Access Journals (Sweden)

    Williams Sylvain

    2006-03-01

    Full Text Available Abstract Background Several clinical studies suggested that antipsychotic-based medications could ameliorate cognitive functions impaired in certain schizophrenic patients. Accordingly, we investigated the effects of various dopaminergic receptor antagonists – including atypical antipsychotics that are prescribed for the treatment of schizophrenia – in a model of toxicity using cultured hippocampal neurons, the hippocampus being a region of particular relevance to cognition. Results Hippocampal cell death induced by deprivation of growth medium constituents was strongly blocked by drugs including antipsychotics (10-10-10-6 M that display nM affinities for D2 and/or D4 receptors (clozapine, haloperidol, (±-sulpiride, domperidone, clozapine, risperidone, chlorpromazine, (+-butaclamol and L-741,742. These effects were shared by some caspases inhibitors and were not accompanied by inhibition of reactive oxygen species. In contrast, (--raclopride and remoxipride, two drugs that preferentially bind D2 over D4 receptors were ineffective, as well as the selective D3 receptor antagonist U 99194. Interestingly, (--raclopride (10-6 M was able to block the neuroprotective effect of the atypical antipsychotic clozapine (10-6 M. Conclusion Taken together, these data suggest that D2-like receptors, particularly the D4 subtype, mediate the neuroprotective effects of antipsychotic drugs possibly through a ROS-independent, caspase-dependent mechanism.

  6. Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor.

    Science.gov (United States)

    Ilchmann, Anne; Krause, Maren; Heilmann, Monika; Burgdorf, Sven; Vieths, Stefan; Toda, Masako

    2012-05-01

    The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove's modified Dulbecco's medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4(+) T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Effect of Somatic Cell Types and Culture Medium on in vitro Maturation, Fertilization and Early Development Capability of Buffalo Oocytes

    Directory of Open Access Journals (Sweden)

    H. Jamil*, H. A. Samad, N. Rehman, Z. I. Qureshi and L. A. Lodhi

    2011-04-01

    Full Text Available This study was designed to evaluate the efficacy of different somatic cell types and media in supporting in vitro maturation (IVM, in vitro fertilization (IVF and early embryonic development competence of buffalo follicular oocytes. Cumulus oocyte complexes were collected for maturation from follicles (>6mm of buffalo ovaries collected at the local abattoir. Oocytes were co-cultured in tissue culture medium (TCM-199 with either granulosa cells, cumulus cells, or buffalo oviductal epithelial cells (BOEC @ 3x106 cells/ml or in TCM-199 without helper cells (control at 39°C and 5%CO2 in humidified air. Fresh semen was prepared in modified Ca++ free Tyrode medium. Fertilization was carried out in four types of media: i Tyrode lactate albumin pyruvate (TALP, ii TALP+BOEC, iii modified Ca++ free Tyrode and iv modified Ca++ free Tyrode+BOEC. Fertilized oocytes were cultured for early embryonic development in TCM-199 with and without BOEC. Higher maturation rates were observed in the granulosa (84.24% and cumulus cells (83.44% than BOEC co culture system (73.37%. Highest fertilization rate was obtained in modified Ca++ free Tyrode with BOEC co culture (70.42%, followed by modified Ca++ free Tyrode alone (63.77%, TALP with BOEC (36.92% and TALP alone (10.94%. Development of early embryos (8-cell stage improved in TCM-199 with BOEC co culture than TCM-199 alone. From the results of this study, it can be concluded that addition of somatic cells (granulosa cells, cumulus cells results in higher maturation rates of buffalo follicular oocytes than BOEC co culture system, while fertilization rate improved in modified Ca++ free Tyrode with and without BOEC. Addition of BOEC to TCM-199 improved the developmental capacity of early embryo.

  8. Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Nakano, S.

    1979-01-01

    Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various nontoxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequecy was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair. (Auth.)

  9. Uranium(VI) complexation in cell culture medium: influence of speciation on Normal Rat Kidney (NRK-52E) cell accumulation

    International Nuclear Information System (INIS)

    Carriere, M.; Khodja, H.; Avoscan, L.; Carrot, F.; Gouget, B.

    2005-01-01

    Uranium bioavailability and toxicity are closely linked to the metal's speciation in solution. However in biological fluids or in media classically used for cell culture - and subsequently for in vitro cell exposure -, uranium is rarely present as free-ion since these media contain non-negligible concentrations of potential ligands such as phosphate and bicarbonate but also co-ions such as calcium which can cause U(VI) complexes precipitation. The chemical form of uranium that is internalized in cells and interferes with biological processes is of major concern. Uranium toxicity and accumulation were evaluated in vitro on NRK-52 E cells, model for rat renal proximal tubule. Uranium intracellular accumulation begins after 12 h exposure to 600 μM U(VI); toxicity appears as soon as cells accumulated 25 to 30 mg U/g protein. Modification of uranium speciation in the exposure medium induces great changes in toxicity and cell accumulation. Comparison of toxicity and accumulation results to theoretical uranium speciation, calculated with the J-Chess computer program, shows that free-ion concentration can not explain the total uranium intracellular accumulation. Low molecular weight U(VI) complexes, such as UO 2 (CO 3 ) 3 4- but also UO 2 PO 4 - could be implicated in U(VI) cellular accumulation and toxicity. (orig.)

  10. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

    Science.gov (United States)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

  11. Kinetic response of a Drosophila melanogaster cell line to different medium formulations and culture conditions

    OpenAIRE

    Bovo, R.; Galesi, A. L. L; Jorge, S. A. C.; Piccoli, R. A. M.; Moraes, A. M.; Pereira, C. A.; Augusto, E. F. P.

    2008-01-01

    In the past few years, Drosophila melanogaster cells have been employed for recombinant protein production purposes, and a comprehensive knowledge of their metabolism is essential for process optimization. In this work, the kinetic response of a Schneider S2 cell line, grown in shake flasks, in two different culture media, the serum-free SF900-II® and the serum-supplemented TC-100, was evaluated. Cell growth, amino acids and glucose uptake, and lactate synthesis were measured allowing the cal...

  12. Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells

    Czech Academy of Sciences Publication Activity Database

    Kunová, M.; Matulka, K.; Eiselleová, L.; Trčková, P.; Hampl, Aleš; Dvořák, Petr

    2010-01-01

    Roč. 21, - (2010), s. 676-686 ISSN 1472-6483 Grant - others:GA MŠk(CZ) LC06077; EC FP6(XE) LSHG-CT-2006-018739 Program:LC Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : animal protein-free culture * high-density culture * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.285, year: 2010

  13. Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

    International Nuclear Information System (INIS)

    Bernard, Vladan; Skorpikova, Jirina; Mornstein, Vojtech; Fojt, Lukas

    2011-01-01

    The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 Wcm 2 in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were - 1 s drop time, 5 mV.s -1 voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag|AgCl|3 M KCI as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy. (author)

  14. Effects of x-irradiation on cell division, oxygen consumption, and growth medium pH of an insect cell line cultured in vitro

    International Nuclear Information System (INIS)

    Koval, T.M.; Myser, W.C.; Hink, W.F.

    1975-01-01

    Cultured Trichoplusia ni cells in exponential growth were administered x-ray doses of 10,000 R and then subcultured. The untreated cell population began exponential growth within a few hours after subculture, eventually reaching stationary growth phase 96 hr later at a cell density of 2.08 x 10 6 cells/ml, whereas the irradiated cell population did not change for 24 hr after irradiation and then began exponential growth at a rate similar to that of control cells, also reaching stationary phase at 96 hr, but at a cell density of 0.93 x 10 6 cells/ml, which is less than half the maximum density of controls. From 24 to 96 hr after treatment, the x-irradiated cells were characterized by an increased consumption of oxygen that was nearly twice the amount utilized by control cells. The pH of the cell growth medium increases over 96 hr from 6.3 to 6.6 for irradiated as well as for untreated cultures, but since the number of x-rayed cells is less than half the number of untreated cells, the pH increase, per cell, of medium from irradiated cultures is about twice that of medium from control cultures

  15. Validation of the BacT/ALERT®3D automated culture system for the detection of microbial contamination of epithelial cell culture medium.

    Science.gov (United States)

    Plantamura, E; Huyghe, G; Panterne, B; Delesalle, N; Thépot, A; Reverdy, M E; Damour, O; Auxenfans, Céline

    2012-08-01

    Living tissue engineering for regenerative therapy cannot withstand the usual pharmacopoeia methods of purification and terminal sterilization. Consequently, these products must be manufactured under aseptic conditions at microbiologically controlled environment facilities. This study was proposed to validate BacT/ALERT(®)3D automated culture system for microbiological control of epithelial cell culture medium (ECCM). Suspensions of the nine microorganisms recommended by the European Pharmacopoeia (Chap. 2.6.27: "Microbiological control of cellular products"), plus one species from oral mucosa and two negative controls with no microorganisms were prepared in ECCM. They were inoculated in FA (anaerobic) and SN (aerobic) culture bottles (Biomérieux, Lyon, France) and incubated in a BacT/ALERT(®)3D automated culture system. For each species, five sets of bottles were inoculated for reproducibility testing: one sample was incubated at the French Health Products Agency laboratory (reference) and the four others at Cell and Tissue Bank of Lyon, France. The specificity of the positive culture bottles was verified by Gram staining and then subcultured to identify the microorganism grown. The BacT/ALERT(®)3D system detected all the inoculated microorganisms in less than 2 days except Propionibacterium acnes which was detected in 3 days. In conclusion, this study demonstrates that the BacT/ALERT(®)3D system can detect both aerobic and anaerobic bacterial and fungal contamination of an epithelial cell culture medium consistent with the European Pharmacopoeia chapter 2.6.27 recommendations. It showed the specificity, sensitivity, and precision of the BacT/ALERT(®)3D method, since all the microorganisms seeded were detected in both sites and the uncontaminated medium ECCM remained negative at 7 days.

  16. Ultrastructure of single cells, callus-like and monospore-like cells in Porphyra yezoensis ueda on semisolid culture medium

    Science.gov (United States)

    Mei, Junxue; Shen, Songdong; Jiang, Ming; Fei, Xiugeng

    2003-06-01

    It had been demonstrated that individual cells or protoplasts isolated from Porphyra thallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi-solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi-solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi-solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus-like cells formed on semi-solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2-layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.

  17. A pumpless cell culture chip with the constant medium perfusion-rate maintained by balanced droplet dispensing.

    Science.gov (United States)

    Kim, Taeyoon; Cho, Young-Ho

    2011-05-21

    This paper presents a pumpless cell culture chip, where a constant-rate medium perfusion is achieved by balanced droplet dispensing. Previous pumpless cell culture chips, where the gravity-driven flow is induced by gradually decreasing the hydraulic-head difference, Δh, between source and drain reservoirs, result in a decreasing perfusion-rate. However, the present pumpless cell culture chip, where autonomous droplet dispensers are integrated on the source reservoirs, results in a constant perfusion-rate using a constant Δh maintained by balanced droplet dispensing between the source-inlet and the drain-outlet. In the experimental study, constant perfusion-rates of 0.1, 0.2, and 0.3 μl min(-1) are obtained by Δh of 38, 76, and 114 mm, respectively. At the constant perfusion-rate (Q=0.2 μl min(-1)), H358 lung cancer cells show the maximum growth-rate of 57.8 ± 21.1% d(-1), which is 1.9 times higher than the 30.2 ± 10.3% d(-1) of the static culture. At a perfusion-rate varying between 0.1-0.3 μl min(-1) (average=0.2 μl min(-1)), however, the H358 cells show a growth-rate of 46.9 ± 8.3% d(-1), which is lower than that of the constant Q of 0.2 μl min(-1). The constant-rate perfusion culture (Q=0.1, 0.2, and 0.3 μl min(-1)) also results in an average cell viability of 89.2%, which is higher than 75.9% of the static culture. This pumpless cell culture chip offers a favorable environment to cells with a high growth-rate and viability, thus having potential for use in cell-based bio-assays. © The Royal Society of Chemistry 2011

  18. Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

    DEFF Research Database (Denmark)

    Liberski, A. R.; Al-Noubi, M. N.; Rahman, Z. H.

    2013-01-01

    rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino......Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only...... developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein...

  19. Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950.

    Science.gov (United States)

    Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna

    2015-01-01

    Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g) were found in yeast cells after 24-hour culture conducted in control (YPD) medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours) the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium) of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

  20. Influence of Selenium Content in the Culture Medium on Protein Profile of Yeast Cells Candida utilis ATCC 9950

    Directory of Open Access Journals (Sweden)

    Marek Kieliszek

    2015-01-01

    Full Text Available Selenium is an essential trace element for human health and it has been recognized as a component of several selenoproteins with crucial biological functions. It has been identified as a component of active centers of many enzymes, as well as integral part of biologically active complexes. The aim of the study was to evaluate the protein content and amino acid profile of the protein of fodder yeast Candida utilis ATCC 9950 cultured in media control and experimental enriched selenium. Protein analysis was performed using SDS-PAGE method consisting of polyacrylamide gel electrophoresis in the presence of SDS. The highest contents of soluble protein (49,5 mg/g were found in yeast cells after 24-hour culture conducted in control (YPD medium. In the presence of selenium there were determined small amounts of protein content. With increasing time of yeast culture (to 72 hours the control and experimental media were reported to reduce soluble protein content. In electropherogram proteins from control cultures was observed the presence of 10 protein fractions, but in all the experimental cultures (containing 20, 30, and 40 mg/L selenium of 14 protein fractions. On the basis of the molecular weights of proteins, it can be concluded that they were among others: selenoprotein 15 kDa and selenoprotein 18 kDa.

  1. Single Cell Protein Production by Saccharomyces cerevisiae Using an Optimized Culture Medium Composition in a Batch Submerged Bioprocess.

    Science.gov (United States)

    Hezarjaribi, Mehrnoosh; Ardestani, Fatemeh; Ghorbani, Hamid Reza

    2016-08-01

    Saccharomyces cerevisiae PTCC5269 growth was evaluated to specify an optimum culture medium to reach the highest protein production. Experiment design was conducted using a fraction of the full factorial methodology, and signal to noise ratio was used for results analysis. Maximum cell of 8.84 log (CFU/mL) was resulted using optimized culture composed of 0.3, 0.15, 1, and 50 g L(-1) of ammonium sulfate, iron sulfate, glycine, and glucose, respectively at 300 rpm and 35 °C. Glycine concentration (39.32 % contribution) and glucose concentration (36.15 % contribution) were determined as the most effective factors on the biomass production, while Saccharomyces cerevisiae growth had showed the least dependence on ammonium sulfate (5.2 % contribution) and iron sulfate (19.28 % contribution). The most interaction was diagnosed between ammonium sulfate and iron sulfate concentrations with interaction severity index of 50.71 %, while the less one recorded for glycine and glucose concentration was equal to 8.12 %. An acceptable consistency of 84.26 % was obtained between optimum theoretical cell numbers determined by software of 8.91 log (CFU/mL), and experimentally measured one at optimal condition confirms the suitability of the applied method. High protein content of 44.6 % using optimum culture suggests that Saccharomyces cerevisiae is a good commercial case for single cell protein production.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  3. Brain stem slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture.

    Science.gov (United States)

    Kaiser, Andreas; Kale, Ajay; Novozhilova, Ekaterina; Siratirakun, Piyaporn; Aquino, Jorge B; Thonabulsombat, Charoensri; Ernfors, Patrik; Olivius, Petri

    2014-05-30

    Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. An absolute procedure to test the growth potential of medium and the influence of decreased oxygen tension in primary amniotic fluid cell cultures

    NARCIS (Netherlands)

    Sikkema-Raddatz, Birgit; Suijkerbuijk, Ron; van der Vlag, Jakob; Stoepker, Marian; Buys, Charles H. C. M.; Meerman, Gerard J. te

    Objective For prenatal cytogenetic diagnosis, cell cultures should be maximally successful. When introducing a change in conditions, e.g. a new batch of medium, the growth potential of a culture is usually compared under both the new condition and the one already in use. Such a relative test is in

  5. An absolute procedure to test the growth potential of medium and the influence of decreased oxygen tension in primary amniotic fluid cell cultures.

    NARCIS (Netherlands)

    Sikkema-Raddatz, B.; Suijkerbuijk, R.F.; Vlag, J. van der; Stoepker, M.; Buys, C.H.C.M.; Meerman, G.J. te

    2006-01-01

    OBJECTIVE: For prenatal cytogenetic diagnosis, cell cultures should be maximally successful. When introducing a change in conditions, e.g. a new batch of medium, the growth potential of a culture is usually compared under both the new condition and the one already in use. Such a relative test is in

  6. AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration

    NARCIS (Netherlands)

    Adam, Aziza A. A.; van Wenum, Martien; van der Mark, Vincent A.; Jongejan, Aldo; Moerland, Perry D.; Houtkooper, Riekelt H.; Wanders, Ronald J. A.; Oude Elferink, Ronald P.; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

    2017-01-01

    Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we

  7. Cytotoxicity of cancer HeLa cells sensitivity to normal MCF10A cells in cultivations with cell culture medium treated by microwave-excited atmospheric pressure plasmas

    Science.gov (United States)

    Takahashi, Yohei; Taki, Yusuke; Takeda, Keigo; Hashizume, Hiroshi; Tanaka, Hiromasa; Ishikawa, Kenji; Hori, Masaru

    2018-03-01

    Cytotoxic effects of human epithelial carcinoma HeLa cells sensitivity to human mammary epithelial MCF10A cells appeared in incubation with the plasma-activated medium (PAM), where the cell culture media were irradiated with the hollow-shaped contact of a continuously discharged plasma that was sustained by application of a microwave power under Ar gas flow at atmospheric pressure. The discharged plasma had an electron density of 7  ×  1014 cm-3. As the nozzle exit to the plasma source was a distance of 5 mm to the medium, concentrations of 180 µM for H2O2 and 77 µM for NO2- were generated in the PAM for 30 s irradiation, resulting in the control of irradiation periods for aqueous H2O2 with a generation rate of 6.0 µM s-1, and nitrite ion (NO2- ) with a rate of 2.2 µM s-1. Effective concentrations of H2O2 and NO2- for the antitumor effects were revealed in the microwave-excited PAM, with consideration of the complicated reactions at the plasma-liquid interfaces.

  8. Size distribution of fullerenol nanoparticles in cell culture medium and their influence on antioxidative enzymes in Chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Srđenović Branislava U.

    2015-01-01

    Full Text Available Fullerenol (C60(OH24 nanoparticles (FNP have a significant role in biomedical research due to their numerous biological activities, some of which are cytoprotective and antioxidative properties. The aim of this study was to measure distribution of fullerenol nanoparticles and zeta potential in cell medium RPMI 1640 with 10% fetal bovine serum (FBS and to investigate the influence of FNP on Chinese hamster ovary cells (CHO-K1 survival, as well as to determine the activity of three antioxidative enzymes: superoxide-dismutase, glutathione-reductase and glutathione-S-transferase in mitomycin C-treated cell line. Our investigation implies that FNP, as a strong antioxidant, influence the cellular redox state and enzyme activities and thus may reduce cell proliferation, which confirms that FNP could be exploited for its use as a cytoprotective agent.[Projekat Ministarstva nauke Republike Srbije, br. III45005 i Pokrajinski Sekretarijat za nauku i tehnološki razvoj Vojvodine, grant number 114-451-2056/2011-01

  9. Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.

    Science.gov (United States)

    Schubert, Susanna; Brehm, Walter; Hillmann, Aline; Burk, Janina

    2018-01-01

    For clinical applications of multipotent mesenchymal stromal cells (MSCs), serum-free culture is preferable to standardize cell products and prevent contamination with pathogens. In contrast to human MSCs, knowledge on serum-free culture of large animal MSCs is limited, despite its relevance for preclinical studies and development of veterinary cellular therapeutics. This study aimed to evaluate the suitability of a commercially available serum-free human MSC medium for culturing equine adipose-derived MSCs in comparison with human adipose MSCs. Enzyme-free isolation by explant technique and expansion of equine and human cells in the serum-free medium were feasible. However, serum-free culture altered the morphology and complicated handling of equine MSCs, with cell aggregation and spontaneous detachment of multilayers, compared to culture in standard medium supplemented with fetal bovine serum. Furthermore, proliferation and the surface immunophenotype of equine cells were more variable compared to the controls and appeared to depend on the lot of the serum-free medium. Particularly the expression of CD90 was different between experimental groups (P cultured in serum-free medium (5.21-83.40%) compared to standard medium (86.20-99.50%). Additionally, small subpopulations expressing MSC exclusion markers such as CD14 (0.28-11.60%), CD34 (0.00-9.87%), CD45 (0.35-10.50%), or MHCII (0.00-3.67%) were found in equine samples after serum-free culture. In contrast, human samples displayed a more consistent morphology and a consistent CD29 + (98.60-99.90%), CD73 + (94.60-98.40%), CD90 + (99.60-99.90%), and CD105 + (97.40-99.80%) immunophenotype after culture in serum-free medium. The obtained data demonstrate that the serum-free medium was suitable for human MSC culture but did not lead to entirely satisfactory results in equine MSCs. This underlines that requirements regarding serum-free culture conditions are species-specific, indicating a need for serum-free media to

  10. Conditioned Medium of Wharton's Jelly Derived Stem Cells Can Enhance the Cartilage Specific Genes Expression by Chondrocytes in Monolayer and Mass Culture Systems

    Directory of Open Access Journals (Sweden)

    Maryam Hassan Famian

    2017-04-01

    Full Text Available Purpose: Mesenchymal stem cells (MSCs have been introduced for cell therapy strategies in osteoarthritis (OA. Despite of their capacity for differentiation into chondrocyte, there are some evidences about their life-threatening problem after transplantation. So, some researchers shifted on the application of stem cells conditioned medium. The goal of this study is to evaluate whether Wharton's jelly derived stem cell conditioned medium (WJSCs-CM can enhance the gene expression profile by chondrocytes in monolayer and mass culture systems. Methods: Conditioned medium was obtained from WJSCs at fourth passage. Isolated chondrocytes were plated at density of 1×106 for both monolayer and high density culture. Then cells in both groups were divided into control (received medium and experiment group treated with WJ-CM for 3 and 6 days. Samples were prepared to evaluate gene expression profile of collagen II, aggrecan, cartilage oligomeric matrix protein (COMP and sox-9 using real-time RT-PCR. Results: After 3 days, Chondrocytes treated with WJSCs-CM expressed significantly higher level of genes compared to the control group in both culture systems. After 6 days, the expression of genes in monolayer cultivated chondrocytes was decreased but that of the mass culture were up-regulated significantly. Conclusion: WJ-SCs-CM can increase the expression of cartilage-specific genes and can be introduced as a promoting factor for cartilage regeneration.

  11. Conditioned medium from MCF-7 cell line induces myofibroblast differentiation, decreased cell proliferation, and increased apoptosis in cultured normal fibroblasts but not in fibroblasts from malignant breast tissue.

    Science.gov (United States)

    Valenti, M T; Azzarello, G; Balducci, E; Sartore, S; Sandri, M; Manconi, R; Sicari, U; Bari, M; Vinante, O

    2001-01-01

    We studied the effect of conditioned medium (CM) obtained from cultures of oestrogen-receptor positive breast cancer MCF7 cell line on the differentiation, proliferation and apoptosis patterns of cultured breast fibroblasts from normal interstitial and malignant stromal tissue. Fibroblasts were grown in the presence or absence of CM and examined for the differentiation pattern by immunofluorescence and Western blotting procedures, for proliferation profile by Ki67 expression, and for apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling technique. Monoclonal antibodies specific for non-muscle (NM), smooth muscle (SM) lineage and differentiation markers were applied to these cultures. CM is able to induce a SM-like differentiation in interstitial fibroblasts, i.e., essentially myofibroblast formation. Fibroblasts from tumour stroma showed the presence of a small number of smooth muscle cells (SMC) along with a large number of myofibroblasts. Treatment of these cultures with CM was unable to change this pattern. Only normal fibroblasts were responsive to the proliferation/apoptotic-inhibitory effect of the CM. These data suggest that structural and functional differences exist between stromal fibroblasts from normal breast and breast cancer with respect to the responsiveness to soluble factors present in the CM. We hypothesize that the lack of in vitro sensitivity to CM shown by 'tumour' fibroblasts is the result of an in vivo inherent and stable phenotypic change on the fibroblasts surrounding breast tumour cells occurring via a paracrine mechanism.

  12. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

    International Nuclear Information System (INIS)

    Yoshito, Daniele

    2011-01-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

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

    Science.gov (United States)

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

    2015-11-01

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

  14. Construction of three-dimensional liver tissue models by cell accumulation technique and maintaining their metabolic functions for long-term culture without medium change.

    Science.gov (United States)

    Matsuzawa, Atsushi; Matsusaki, Michiya; Akashi, Mitsuru

    2015-04-01

    Three-dimensional (3D) hepatocyte cultures have attracted much attention to obtain high biological functions of hepatocyte for pharmaceutical drug assessment. However, maintaining the high functions for over one month is still a key challenge although many approaches have been reported. In this study, we demonstrate for the first time simple and rapid construction of 3D-hepatocyte constructs by our cell accumulation technique and their high biological functions for one month, without any medium change. The human hepatocyte carcinoma (HepG2) cells were coated with ∼ 7 nm-sized extracellular matrix (ECM) films consisting of fibronectin (FN) and gelatin (G), and then incubated in cell culture insert to construct 3D-tissue constructs for 24 h. The thickness of obtained 3D-HepG2 constructs was easily controlled by altering seeding cell number and the maximum is over 100 μm. When a large volume of culture media was employed, the 3D-constructs showed higher mRNA expression of albumin and some cytochrome P450 (CYP) enzymes as compared to general two-dimensional (2D) culture. Surprisingly, their high cell viabilities (over 80%) and high mRNA expressions were successfully maintained without medium change for at least 27 days. These results demonstrate novel easy and rapid technique to construct 3D-human liver tissue models which can maintain their high functions and viability for 1 month without medium change. © 2014 Wiley Periodicals, Inc.

  15. Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

    International Nuclear Information System (INIS)

    Sandru, G.; Greiner, R.

    1994-01-01

    Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

  16. 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...

  17. The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3

    International Nuclear Information System (INIS)

    Alper, O.; Yamaguchi, K.; Hitomi, J.; Honda, S.; Matsushima, T.; Abe, K.

    1990-01-01

    The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein

  18. Altered cytotoxicity of ROS-inducing compounds by sodium pyruvate in cell culture medium depends on the location of ROS generation.

    Science.gov (United States)

    Kelts, Jessica L; Cali, James J; Duellman, Sarah J; Shultz, John

    2015-01-01

    Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol >10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra- or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

  19. Optimization of culture medium for novel cell-associated tannase production from Bacillus massiliensis using response surface methodology.

    Science.gov (United States)

    Belur, Prasanna D; Goud, Rakesh; Goudar, Dinesh C

    2012-02-01

    Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2(3) factorial design augmented by 7 axial points (alpha = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30 degrees C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid.

  20. Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture.

    Science.gov (United States)

    Oze, Hiroki; Hirao, Makoto; Ebina, Kosuke; Shi, Kenrin; Kawato, Yoshitaka; Kaneshiro, Shoichi; Yoshikawa, Hideki; Hashimoto, Jun

    2012-02-01

    Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O(2) (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm(2) flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O(2) in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O(2), respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O(2) and 500 μl (1,480-μm depth) at 5% O(2) in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.

  1. A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

    Science.gov (United States)

    Ahmadian Baghbaderani, Behnam; Tian, Xinghui; Scotty Cadet, Jean; Shah, Kevan; Walde, Amy; Tran, Huan; Kovarcik, Don Paul; Clarke, Diana; Fellner, Thomas

    2016-01-01

    Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

  2. Detection and characterization of silver nanoparticles and dissolved species of silver in culture medium and cells by AsFlFFF-UV-Vis-ICPMS: application to nanotoxicity tests.

    Science.gov (United States)

    Bolea, E; Jiménez-Lamana, J; Laborda, F; Abad-Álvaro, I; Bladé, C; Arola, L; Castillo, J R

    2014-03-07

    A methodology based on Asymmetric Flow Field-Flow Fractionation (AsFlFFF) coupled with UV-Vis absorption spectrometry and ICP mass spectrometry (ICPMS) has been developed and applied to the study of silver nanoparticles (AgNPs) and dissolved species of silver in culture media and cells used in cytotoxicity tests. The effect of a nano-silver based product (protein stabilized silver nanoparticles ca. 15 nm average diameter) on human hepatoma (HepG2) cell viability has been studied. UV-Vis absorption spectrometry provided information about the nature (organic vs. nanoparticle) of the eluted species, whereas the silver was monitored by ICPMS. A shift towards larger hydrodynamic diameters was observed in the AgNPs after a 24 hour incubation period in the culture medium, which suggests a "protein corona" effect. Silver(I) associated with proteins present in the culture medium has also been detected, as a consequence of the oxidation process experimented by the AgNPs. However, the Ag(I) released into the culture medium did not justify the toxicity levels observed. AgNPs associated with the cultured HepG2 cells were also identified by AsFlFFF, after applying a solubilisation process based on the use of tetramethylammonium hydroxide (TMAH) and Triton X-100. These results have been confirmed by transmission electronic microscopy (TEM) analysis of the fractions collected from the AsFlFFF. The effect of AgNPs on HepG2 cells has been compared to that caused by silver(I) as AgNO3 under the same conditions. The determination of the total content of silver in the cells confirms that a much larger mass of silver as AgNPs with respect to AgNO3 (16 to 1) is needed to observe a similar toxicity.

  3. Human umbilical cord stem cell conditioned medium versus serum-free culture medium in the treatment of cryopreserved human ovarian tissues in in-vitro culture: a randomized controlled trial.

    Science.gov (United States)

    Jia, Yingxian; Shi, Xiaohan; Xie, Yidong; Xie, Xiaochuan; Wang, Yan; Li, Shangwei

    2017-06-24

    To reduce young female fertility loss, the in-vitro culture of cryopreserved ovarian cortical tissues (OCTs) is considered an effective approach without delaying treatment and undergoing stimulation medicine. However, ischemic damage and follicular loss during the in-vitro culture of OCTs are major technical challenges. Human umbilical cord stem cells (HUMSCs) and their conditioned medium (HUMSC-CM) have been considered to be potential resources for regeneration medicine because they secrete cytokines and enhance cell survival and function. The aim of this study was to determine whether HUMSC-CM improves the development of frozen-thawed in-vitro cultured ovarian tissues compared with a serum-free culture medium (SF-CM). The thawed OCTs (n = 68) were cultivated in HUMSC-CM and SF-CM in vitro for 8 days, and the ovarian tissues were processed and analyzed by a classical histological evaluation. The microvessel density (MVD) and apotosis detection during in-vitro culture of OCTs were also performed. A significant difference in the rate of morphologically normal primordial follicles in the HUMSC-CM group was observed compared to that in the SF-CM group (group C) from days 2 to 4 (day 2: group B 58.0 ± 2.45% vs group C 32.0 ± 5.83%, p = 0.002; day 3: group B 55.5 ± 4.20% vs group C 21.0 ± 9.80%, p = 0.048; day 4: group B 52.0 ± 4.08% vs group C 21.5 ± 8.19%, p = 0.019). The microvessel density (MVD) detection showed a time-dependent increase and peaked on day 4. There was a significant difference between groups B (49.33 ± 0.58) and C (24.33 ± 3.79) (p = 0.036). The percentage of apoptotic follicles in group B was lower than that in group C on day 1 (13.75 ± 2.50% vs 27.0 ± 10.10%, p = 0.003), day 5 (11.75 ± 1.50% vs 51.0 ± 10.5%, p = 0.019) and day 7 (15.0 ± 5.10% vs 46.5 ± 21.75%, p = 0.018). These data have provided the first experimental evidence of the effect of

  4. Evaluation of Insulin Medium or Chondrogenic Medium on Proliferation and Chondrogenesis of ATDC5 Cells

    OpenAIRE

    Yao, Yongchang; Zhai, Zhichen; Wang, Yingjun

    2014-01-01

    Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured ...

  5. Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

    Directory of Open Access Journals (Sweden)

    Minoru Tomizawa

    Full Text Available Hepatocyte differentiation inducer (HDI lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE medium, followed by a 2-day culture in HDI.Expression levels of α-feto protein (AFP were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12. 201B7 cells expressed the highest AFP and albumin (ALB when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition.201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.

  6. Selective medium for culture of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Cook, Beth S; Beddow, Jessica G; Manso-Silván, Lucía; Maglennon, Gareth A; Rycroft, Andrew N

    2016-11-15

    The fastidious porcine respiratory pathogen Mycoplasma hyopneumoniae has proven difficult to culture since it was first isolated in 1965. A reliable solid medium has been particularly challenging. Moreover, clinical and pathological samples often contain the fast-growing M. hyorhinis which contaminates and overgrows M. hyopneumoniae in primary culture. The aim of this study was to optimise the culture medium for recovery of M. hyopneumoniae and to devise a medium for selection of M. hyopneumoniae from clinical samples also containing M. hyorhinis. The solid medium devised by Niels Friis was improved by use of Purified agar and incorporation of DEAE-dextran. Addition of glucose or neutralization of acidity in liquid medium with NaOH did not improve the final yield of viable organisms or alter the timing of peak viability. Analysis of the relative susceptibility of M. hyopneumoniae and M. hyorhinis strains to four antimicrobials showed that M. hyopneumoniae is less susceptible than M. hyorhinis to kanamycin. This was consistent in all UK and Danish strains tested. A concentration of 2μg/ml of kanamycin selectively inhibited the growth of all M. hyorhinis tested, while M. hyopneumoniae was able to grow. This forms the basis of an effective selective culture medium for M. hyopneumoniae. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Adding of ascorbic acid to the culture medium influences the antioxidant status and some biochemical parameters in the hen granulosa cells.

    Science.gov (United States)

    Capcarova, M; Kolesarova, A; Kalafova, A; Bulla, J; Sirotkin, A V

    2015-07-01

    The aim of the present study was to determine the activity of superoxide dismutase (SOD), total antioxidant status (TAS) of the hen granulosa cells, and selected biochemical parameters, including calcium, phosphorus, sodium, potassium, glucose, cholesterol, proteins, in the culture medium of granulosa cells after exposing them to ascorbic acid in vitro conditions. Ovarian granulosa cells of hens were incubated with various doses of ascorbic acid (E1 0.09 mg/ml, E2 0.13 mg/ml, E3 0.17 mg/ml, E4 0.33 mg/ml, E5 0.5 mg/ml). Ascorbic acid did not manifest antioxidant potential and higher doses of ascorbic acid (0.17; 0.33 and 0.5 mg/ml) decreased the activity of SOD in granulosa cells. Vitamin application resulted in a significantly (pascorbic acid might be involved in the regulation of selected biochemical and physiological processes in ovarian granulosa cells.

  8. 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....

  9. Novel culture medium for the axenic growth of Balamuthia mandrillaris.

    Science.gov (United States)

    Lares-Jiménez, Luis Fernando; Gámez-Gutiérrez, Ricardo Alfredo; Lares-Villa, Fernando

    2015-08-01

    Until now, for axenic cultivation of Balamuthia mandrillaris, the BM-3 culture medium and the Modified Chang's special medium have been the only ones recommended, but they have some disadvantages, as both require many components and their preparations are laborious. Therefore, we developed a novel culture medium for B. mandrillaris axenic cultivation. Each one of the 11 components of BM-3 was combined with Cerva's medium as basal culture medium. Ten strains of B. mandrillaris including the reference strain CDC:V039 and 9 environmental isolates were used during trials. After testing all combinations, the basal medium complemented with 10× Hank's balanced salt solution was the only one that supported confluent growth of B. mandrillaris. Cell shape and motility of trophozoites were normal. This developed medium is as useful as BM-3 for axenization. The development of a cheaper and easy-to-prepare medium for B. mandrillaris opens the possibility of increasing its study. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Correlation between culture medium pH, extracellular proteinase activity, and cell growth of Candida albicans in insoluble stratum corneum-supplemented media.

    Science.gov (United States)

    Tsuboi, R; Matsuda, K; Ko, I J; Ogawa, H

    1989-01-01

    Candida albicans produces a major extracellular proteinase whose activities are observed only in weakly acidic pH. However, in affected lesions, a variety of pH conditions exist, including neutral pH. To verify the pathological importance of the extracellular proteinase, the correlation between culture medium pH, extracellular proteinase activity, and cell growth of C. albicans was followed for 3 weeks with unbuffered and insoluble stratum corneum-supplemented liquid media. Each medium pH, initially adjusted within a range of pH 3-7 by the addition of sodium hydroxide or hydrochloric acid solution, was acidified, and a subsequent high proteolytic activity and rapid fungal growth were observed. After full fungal growth, neutralization of each medium to pH 7 and reduction of proteinase activity occurred. Results from a glucose addition experiment suggest that acidification of each medium was produced by the acid formation from glucose and neutralization by the exhaustion of glucose and increase of ammonia from denatured stratum corneum. These data suggest that extracellular proteinase from C. albicans could act as a virulence factor under a wide range of pH conditions by the acidification of the environmental pH close to the organism.

  11. Drug and radiation sensitivity measurements of successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and supplemented medium

    International Nuclear Information System (INIS)

    Baker, F.L.; Spitzer, G.; Ajani, J.A.

    1986-01-01

    The limitations of the agar suspension culture method for primary culturing of human tumor cells prompted development of a monolayer system optimized for cell adhesion and growth. This method grew 83% of fresh human tumor cell biopsy specimens, cultured and not contaminated, from a heterogeneous group of 396 tumors including lung cancer (93 of 114, 82%); melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a miscellaneous group consisting of gastrointestinal, genitourinary, mesothelioma, and unknown primaries (78 of 91, 86%). Cell growth was characterized morphologically with Papanicolaoustained coverslip cultures and cytogenetically with Giemsastained metaphase spreads. Morphological features such as nuclear pleomorphism, chromatin condensation, basophilic cytoplasm, and melanin pigmentation were routinely seen. Aneuploid metaphases were seen in 90% of evaluable cultures, with 15 of 28 showing 70% or more aneuploid metaphases. Colony-forming efficiency ranged between 0.01 and 1% of viable tumor cells, with a median efficiency of 0.2%. This culture system uses a low inoculum of 25,000 viable cells per well which permitted chemosensitivity testing of nine drugs at four doses in duplicate from 2.2 X 10(6) viable tumor cells and radiation sensitivity testing at five doses in quadruplicate from 0.6 X 10(6) cells. Cultures were analyzed for survival by computerized image analysis of crystal violet-stained cells. Drug sensitivity studies showed variability in sensitivity and in survival curve shape with exponential cell killing for cisplatin, Adriamycin, and etoposide, and shouldered survival curves for 5-fluorouracil frequently seen. Radiation sensitivity studies also showed variability in both sensitivity and survival curve shape. Many cultures showed exponential cell killing, although others had shouldered survival curves

  12. Addition of granulosa cell mass to the culture medium of oocytes derived from early antral follicles increases oocyte growth, ATP content, and acetylation of H4K12.

    Science.gov (United States)

    Sugiyama, Miyako; Sumiya, Mei; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

    2016-12-01

    The main aim of the present study was to examine the hypothesis that an increase in the number of granulosa cells surrounding developing bovine oocytes results in both high ATP levels and an increase in the acetylation level of H4K12 in oocytes grown in vitro. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles (EAFs, 0.4-0.7 mm in diameter), and individually cultured on 96-well plates with or without additional granulosa cell mass that had been prepared from other OGCs. After 16 days of culture, we examined: (i) the rate of antrum formation of the OGCs; (ii) the diameter, maturation, and fertilization rate of the oocytes; and (iii) the ATP content and acetylation level of H4K12 in the oocytes grown in vitro. Granulosa cell mass added to the culture medium contributed to the development of OGCs with a higher rate of antrum formation and oocyte growth. Furthermore, the addition of granulosa cells increased the ATP content and acetylation level of H4K12 in oocytes grown in vitro compared with those developed without addition of granulosa cells. In addition, there was a positive correlation between the ATP content in oocytes grown in vitro and the number of granulosa cells in the corresponding OGCs. The results suggest that granulosa cells play a role not only in the development of OGCs and the growth of oocytes, but also in the determination of ATP content and the acetylation of H4K12 in the oocytes developed in vitro.

  13. Influence of serum extraction from the culture medium and of sublethal X-ray irradiation upon microvilli and invaginations of the membrane of Ehrlich ascites tumor cells in monolayer culture

    Energy Technology Data Exchange (ETDEWEB)

    Laudenbach, G.; Pfab, R.; Hess, F.; Schachtschabel, D.O.

    1984-09-01

    In order to find out modifications of microvilli and invaginations, the cellular surfaces of Ehrlich ascites tumor cells in monolayer culture (basal medium of Eagle + 10% fetal calf serum) were investigated with the aid of electron-microscopic cross-sections. The tumor cells had been cultured without serum 24 hours prior to investigation or irradiated with 2 Gy. Morphometric evaluation after cell culture in a serum-free medium showed a reduced number of microvilli and a diminution of sections of microvilli. As already described before, a reduction of cell proliferation, of the microtubule-microfilament system, and of the endocytosis activity occurs under these serum-free conditions. The number of invaginations (related to a constant membrane part) was reduced by nearly 50% after serum extraction. Similarly to serum extraction, sublethal X-ray irradiation reduced the sections of microvilli, whereas the number of microvilli increased slightly. Contrary to the effect of serum extraction, the irradiated cells showed twice as many invaginations as the non-irradiated control cells. These differences in the surface structures are interpreted as a result of modified growth stimulations (+- serum) and radiogenic reparation processes.

  14. Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study

    Energy Technology Data Exchange (ETDEWEB)

    Shchukarev, Andrey, E-mail: andrey.shchukarev@umu.se [Department of Chemistry, Umeå University, Umeå SE-90187 (Sweden); Malekzadeh, Behnosh Öhrnell [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Ransjö, Maria [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Tengvall, Pentti [Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden); Westerlund, Anna [Department of Orthodontics, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-40530 (Sweden)

    2017-04-15

    Highlights: • In the absence of FBS, chemically immobilized insulin layer remains intact; • The immobilized insulin expose hydrophobic domains outward the implant; • In the presence of FBS, a partial replacement of insulin occurs; • The immobilized insulin stabilizes the secondary structure of adsorbed proteins. - Abstract: Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – α-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation.

  15. Surface characterization of insulin-coated Ti6Al4V medical implants conditioned in cell culture medium: An XPS study

    International Nuclear Information System (INIS)

    Shchukarev, Andrey; Malekzadeh, Behnosh Öhrnell; Ransjö, Maria; Tengvall, Pentti; Westerlund, Anna

    2017-01-01

    Highlights: • In the absence of FBS, chemically immobilized insulin layer remains intact; • The immobilized insulin expose hydrophobic domains outward the implant; • In the presence of FBS, a partial replacement of insulin occurs; • The immobilized insulin stabilizes the secondary structure of adsorbed proteins. - Abstract: Surface characterization of insulin-coated Ti6Al4V medical implants, after incubation in α-minimum essential medium (α-MEM), was done by X-ray photoelectron spectroscopy (XPS), in order to analyze the insulin behavior at the implant – α-MEM interface. In the absence of serum proteins in cell culture medium, the coated insulin layer remained intact, but experienced a time-dependent structural transformation exposing hydrophobic parts of the protein toward the solution. The presence of fetal bovine serum (FBS) in the medium resulted in partial substitution of insulin by serum proteins. In spite of some insulin release, the remaining coated layer demonstrated a direct surface effect by stabilizing the structure of protein competitors, and by supporting the accumulation of calcium and phosphate ions at the interface. A structurally stable protein layer with incorporated calcium and phosphate ions at the implant–tissue interface could be an important prerequisite for enhanced bone formation.

  16. Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition.

    Science.gov (United States)

    Liu, Jing; Saponjian, Yero; Mahoney, Mark M; Staley, Kevin J; Berdichevsky, Yevgeny

    2017-01-01

    Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic cultures is driven by these components. We examined the influence of medium composition on epileptogenesis. Epileptogenesis was evaluated by measurements of lactate and lactate dehydrogenase (LDH) levels (biomarkers of ictal activity and cell death, respectively) in spent culture media, immunohistochemistry and automated 3-D cell counts, and extracellular recordings from CA3 regions. Changes in culture medium components moderately influenced lactate and LDH levels as well as electrographic seizure burden and cell death. However, epileptogenesis occurred in any culture medium that was capable of supporting neural survival. We conclude that medium composition is unlikely to be the cause of epileptogenesis in the organotypic hippocampal culture model of chronic post-traumatic epilepsy.

  17. Development of a vinasse culture medium for plant tissue culture

    International Nuclear Information System (INIS)

    Silva, A.L.L.D.; Gollo, L.

    2014-01-01

    Vinasse is the main pollutant (effluent) obtained from the distillation of sugarcane in the production of fuel alcohol. However, this residue is rich in nutrients that are required by plants. We developed a new culture medium using vinasse for the In vitro propagation of an orchid. The vinasse was treated (decanted and filtered), and the nutrients were determined and quantified. Different formulations using vinasse were tested for an In vitro culture. The vinasse dilutions demonstrated a good buffering effect. The ideal vinasse dilution for media formulation was 2.5%. The best KC formulations with vinasse were KCV1 and KCV5. Compared to KC medium, these formulations demonstrated similar results for In vitro multiplication, with the exception of protocorm-like body number, which was inferior in the vinasse formulations. Conversely, for In vitro elongation and rooting, these vinasse media were superior to KC medium. KC medium promotes a low rooting rate (8%) compared to 68 and 100% obtained by KCV1 and KCV5, respectively. Moreover, plantlets cultured on KC medium become protocorm-like body clusters, which impeded the acclimatization of these explants. Plantlets elongated and rooted on KCV1 and KCV5 were successfully acclimatized with a 91% survival rate for both KC vinasse formulations. This study shows the great potential of this technology as a rational alternative to vinasse disposal and adds value to what is currently considered a waste product. (author)

  18. Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2017-01-01

    Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N...

  19. Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

    Czech Academy of Sciences Publication Activity Database

    Bernard, V.; Fojt, Lukáš; Škorpíková, J.; Mornstein, V.

    2011-01-01

    Roč. 48, č. 1 (2011), s. 59-62 ISSN 0301-1208 R&D Projects: GA ČR(CZ) GAP205/10/2378 Keywords : Cisplatin * Differential pulse polarography * Ovarian carcinoma cells A2780 Subject RIV: BO - Biophysics Impact factor: 1.142, year: 2011

  20. Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection.

    Science.gov (United States)

    Erdmann, Michael; Dörrie, Jan; Schaft, Niels; Strasser, Erwin; Hendelmeier, Martin; Kämpgen, Eckhart; Schuler, Gerold; Schuler-Thurner, Beatrice

    2007-09-01

    Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is the current lack of a simple functionally "closed" system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized the use of the Elutra counterflow elutriation system to enrich monocytic DC precursors by (1) developing an algorithm to avoid red blood cell debulking and associated monocyte loss before elutriation, and (2) by elutriation directly in culture medium rather than phosphate-buffered saline. Upon elutriation the bags containing the collected monocytes are simply transferred into the incubator to generate DC progeny as the final "open" washing step is no longer required. Elutriation resulted in significantly more (> or = 2-fold) and purer DC than the standard gradient centrifugation/adherence-based monocyte enrichment, whereas morphology, maturation markers, viability, migratory capacity, and T cell stimulatory capacity were identical. Subsequently, we compared RNA transfection, as this is an increasingly used approach to load DC with antigen. Elutra-derived and adherence-derived DC could be electroporated with similar, high efficiency (on average >85% green fluorescence protein positive), and appeared also equal in antigen expression kinetics. Both Elutra-derived and adherence-derived DC, when loaded with the MelanA peptide or electroporated with MelanA RNA, showed a high T cell stimulation capacity, that is, priming of MelanA-specific CD8+ T cells. Our optimized Elutra-based procedure is straightforward, clearly superior to the standard gradient centrifugation/plastic adherence protocol, and now allows the generation of large numbers of peptide-loaded or RNA-transfected DC in a functionally closed system.

  1. Energy-based culture medium design for biomanufacturing optimization: A case study in monoclonal antibody production by GS-NS0 cells.

    Science.gov (United States)

    Quiroga-Campano, Ana L; Panoskaltsis, Nicki; Mantalaris, Athanasios

    2018-03-02

    Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model's predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies. Copyright © 2018. Published by Elsevier Inc.

  2. INNOVATIVE CULTURE IN SMALL AND MEDIUM ENTERPRISES

    Directory of Open Access Journals (Sweden)

    Aluisio Broering Mambrini

    2011-10-01

    Full Text Available In the last two decades, innovation has been a key driver of economic growth. Innovation is closely related to creating value and generating wealth through successful service to consumer needs. Thus, it is not necessarily restricted to the use of new knowledge generated from research, but on the development of new products or services that are obtained with creative use of knowledge, new or already known. This study aimed to identify management practices that promote a culture of innovation in small and medium enterprises and analyze how they contribute to the innovative capacity of these companies. The research method was the multiple case study with six small and medium businesses that have at least one case of significant innovation in its history. The main results showed that amongst the practices are: a performance in highly specialized niches and deep focus on customer needs; b strong investment and incorporation of new knowledge outside the company (open innovation; c speed and agility in the absorption and deployment of new knowledge and technologies; d retention of employees; e acting as an integrator combining diverse knowledge and technologies; f the information management of the knowledge acquired by the company; g little concern to patent the technology; h flexibility and informal, fluid and open communication between employees of the company that promotes agility in management and i the management of partnerships across the value chain, including the functional areas.

  3. Ureaplasma infection of cell cultures.

    Science.gov (United States)

    Kotani, H; McGarrity, G J

    1986-05-01

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

  4. Medium optimization for protopectinase production by batch culture of

    African Journals Online (AJOL)

    Medium optimization for protopectinase production by batch culture of. C Fan, Z Liu, L Yao. Abstract. Optimization of medium compositions for protopectinase production by Aspergillus terreus in submerged culture was carried out. The medium components having significant effect on protopectinase production were reported ...

  5. Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

    Science.gov (United States)

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Kim, Jin Wook; Lee, Tae Hee; Lee, Byeong Chun

    2015-09-01

    Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P cloned embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Influence of growth factors and medium composition on benzo[a]pyrene- and vitamin A-induced cell proliferation and differentiation in hamster tracheal epithelium in organ culture

    NARCIS (Netherlands)

    Wolterbeek, A.P.M.; Ciotti, M.A.L.T.; Schoevers, E.J.; Roggeband, R.; Baan, R.A.; Feron, V.J.; Rutten, A.A.J.J.L.

    1996-01-01

    Tracheal organ cultures and isolated tracheal epithelial cells are frequently used to study effects of carcinogens and retinoids on both proliferation and differentiation of respiratory tract epithelial cells. For each of these in vitro models, optimal culture conditions have been established,

  7. Buffering the pH of the culture medium does not extend yeast replicative lifespan.

    Science.gov (United States)

    Wasko, Brian M; Carr, Daniel T; Tung, Herman; Doan, Ha; Schurman, Nathan; Neault, Jillian R; Feng, Joey; Lee, Janet; Zipkin, Ben; Mouser, Jacob; Oudanonh, Edward; Nguyen, Tina; Stetina, Torin; Shemorry, Anna; Lemma, Mekedes; Kaeberlein, Matt

    2013-01-01

    During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival.  As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium.  In this study, we assessed whether a similar process occurs during replicative aging of yeast cells.  We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan.  Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.

  8. Immunohistochemical expression of stem cell, endothelial cell, and chemosensitivity markers in primary glioma spheroids cultured in serum-containing and serum-free medium

    DEFF Research Database (Denmark)

    Christensen, Karina; Aaberg-Jessen, Charlotte; Andersen, Claus

    2010-01-01

    To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas....

  9. Long-Term Survival of Leptospira in a Biphasic Culture Medium Containing Charcoal

    Science.gov (United States)

    Myers, Donald M.; Varela-Díaz, Victor M.; Siniuk, Alicia A.

    1973-01-01

    A comparison was made of the survival of 28 leptospiral serotypes in Fletcher semisolid medium and in the same medium containing a basal layer of Fletcher medium plus 0.7% of agar and 0.5% of activated animal charcoal. A year after culture, more motile leptospires were observed by microscope examination in the biphasic medium. Two years after culture, 4 serotypes grown in the biphasic medium and 11 in Fletcher medium did not show motile cells. Nineteen of the serotypes maintained in Fletcher medium and 25 in the biphasic medium for 2 years grew on subculture into Fletcher medium. Subcultures from the biphasic medium showed the characteristic leptospiral ring growth earlier during the incubation period. PMID:4735486

  10. Study on bystander effect and associated mechanism mediated through culture medium

    International Nuclear Information System (INIS)

    Tu Xumin; Lei Suwen; Zhang Zhixing; Lv Huimin

    2005-01-01

    Objective: To study the bystander effect and associated mechanism mediated through the irradiated cell culture medium. Methods: Splenic natural killer (NK) cells were obtained from healthy male ICR strain mice. Culture medium irradiated with different doses of 60 Co γ-rays was used for culturing Yac-I lymphoma cells. The degree of injury of the latter by activated NK cells was observed. A part of the culture media were pretreated with 1% DMSO, a scavenger of reactive oxygen species (ROS), in order to investigate the possible mechanism of a radiation-induced bystander response. Results: Severer injury was induced in Yac-I cells cultured in the media pre-irradiated with different doses of γ-rays than that in Yac-I cells cultured in unirradiated medium, as shown by increased sensitivity to murine splenic NK cells (P<0.01). Culturing Yac-I cells in DMSO-pretreated medium considerably reduced the activation of NK cells, especially in 0.25 Gy and 0.5 Gy γ-irradiated media. Therefore, it can be expected that DMSO can partly suppress ROS-induced bystander effect. Conclusion: The irradiated culture medium of Yac-I cells can trigger bystander effect. ROS likely plays an important role in radiation-induced bystander effect that can be partly suppressed by pretreatment with DMSO. (authors)

  11. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Gisder

    Full Text Available Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin or presumed (surfactin or no (paromomycin activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  12. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Science.gov (United States)

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  13. Human umbilical cord Wharton's jelly stem cells undergo enhanced chondrogenic differentiation when grown on nanofibrous scaffolds and in a sequential two-stage culture medium environment.

    Science.gov (United States)

    Fong, Chui-Yee; Subramanian, Arjunan; Gauthaman, Kalamegam; Venugopal, Jayarama; Biswas, Arijit; Ramakrishna, Seeram; Bongso, Ariff

    2012-03-01

    The current treatments used for osteoarthritis from cartilage damage have their disadvantages of donor site morbidity, complicated surgical interventions and risks of infection and graft rejection. Recent advances in tissue engineering have offered much promise in cartilage repair but the best cell source and in vitro system have not as yet been optimised. Human bone marrow mesenchymal stem cells (hBMSCs) have thus far been the cell of choice. However, we derived a unique stem cell from the human umbilical cord Wharton's jelly (hWJSC) that has properties superior to hBMSCs in terms of ready availability, prolonged stemness characteristics in vitro, high proliferation rates, wide multipotency, non-tumorigenicity and tolerance in allogeneic transplantation. We observed enhanced cell attachment, cell proliferation and chondrogenesis of hWJSCs over hBMSCs when grown on PCL/Collagen nanoscaffolds in the presence of a two-stage sequential complex/chondrogenic medium for 21 days. Improvement of these three parameters were confirmed via inverted optics, field emission scanning electron microscopy (FESEM), MTT assay, pellet diameters, Alcian blue histology and staining, glycosaminglycans (GAG) and hyaluronic acid production and expression of key chondrogenic genes (SOX9, Collagen type II, COMP, FMOD) using immunohistochemistry and real-time polymerase chain reaction (qRT-PCR). In separate experiments we demonstrated that the 16 ng/ml of basic fibroblast growth factor (bFGF) present in the complex medium may have contributed to driving chondrogenesis. We conclude that hWJSCs are an attractive stem cell source for inducing chondrogenesis in vitro when grown on nanoscaffolds and exposed sequentially first to complex medium and then followed by chondrogenic medium.

  14. Palm kernel agar: An alternative culture medium for rapid detection ...

    African Journals Online (AJOL)

    The feasibility of using palm kernel agar (PKA) as an alternative culture medium to desiccated coconut agar (DCA), the conventional medium for the recovery of aflatoxigenic fungi from mixed cultures and the detection of aflatoxigenic fungi and direct visual determination of aflatoxins in agricultural commodities was ...

  15. The culture of Chlorella vulgaris in a recycled supernatant: effects on biomass production and medium quality.

    Science.gov (United States)

    Hadj-Romdhane, F; Zheng, X; Jaouen, P; Pruvost, J; Grizeau, D; Croué, J P; Bourseau, P

    2013-03-01

    Reusing supernatant of microalgae culture medium can have inhibitory or toxic effects on the biomass production because of the release of organic metabolites by cells in the culture medium during their growth. This work investigated the impact of Chlorella vulgaris medium recycling on culture productivity, cells quality and accumulation of excreted metabolites in the culture medium. No significant impact on the C. vulgaris growth was observed after 63days of recycling, the productivity remained stable at around 0.55kgm(-3)day(-1). Organic matters accumulated in supernatant were identified as biopolymers (BP) poor in nitrogen and with a size above 40kDa (probably polysaccharides), and small organic molecules (SOM) richer in nitrogen with a molecular size ranging from 1 to 3kDa. The concentration of biopolymers in the supernatant increased till to a maximum and then decreased, possibly consumed by bacteria, whereas small organic compounds accumulated in the medium. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. The culture of Chlorella vulgaris in a recycled supernatant: Effects on biomass production and medium quality

    KAUST Repository

    Hadj-Romdhane, F.

    2013-03-01

    Reusing supernatant of microalgae culture medium can have inhibitory or toxic effects on the biomass production because of the release of organic metabolites by cells in the culture medium during their growth. This work investigated the impact of Chlorella vulgaris medium recycling on culture productivity, cells quality and accumulation of excreted metabolites in the culture medium. No significant impact on the C. vulgaris growth was observed after 63days of recycling, the productivity remained stable at around 0.55kgm-3day-1. Organic matters accumulated in supernatant were identified as biopolymers (BP) poor in nitrogen and with a size above 40kDa (probably polysaccharides), and small organic molecules (SOM) richer in nitrogen with a molecular size ranging from 1 to 3kDa. The concentration of biopolymers in the supernatant increased till to a maximum and then decreased, possibly consumed by bacteria, whereas small organic compounds accumulated in the medium. © 2013 Elsevier Ltd.

  17. 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.

  18. Methodology for monitoring gold nanoparticles and dissolved gold species in culture medium and cells used for nanotoxicity tests by liquid chromatography hyphenated to inductively coupled plasma-mass spectrometry.

    Science.gov (United States)

    López-Sanz, Sara; Fariñas, Nuria Rodríguez; Vargas, Rosario Serrano; Martín-Doimeadios, Rosa Del Carmen Rodríguez; Ríos, Ángel

    2017-03-01

    An analytical methodology based on coupling reversed-phase liquid chromatography (HPLC) to an inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the characterization and identification of gold nanoparticles (AuNPs) and gold dissolved species (Au 3+ ) in culture medium (Dulbecco's Modified Eagle Medium, DMEM) and HeLa cells (a human cervical adenocarcinoma cell line) used in nanotoxicity tests. The influence of the culture medium was also studied and the method applied for nanotoxicity tests. It was also observed that AuNPs can undergo an oxidation process in the supernatants and only a small amount of AuNPs and dissolved Au 3+ was associated with cells. To evaluate the biological impact of AuNPs, a classical viability assay onto HeLa cells was performed using cellular media DMEM in the presence of increasing dosage of 10nm AuNPs. The results showed that 10nm AuNPs exhibit a slight toxic effect. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Sodium hypochlorite sterilization of culture medium in ...

    African Journals Online (AJOL)

    With the purpose of finding an alternative to thermal sterilization, this research aimed at assessing the efficiency and ideal concentration of sodium hypochlorite for sterilization of culture media and glassware used during rooting of micropropagated Gerbera hybrida cv. Essandre. Two experiments were carried out. In the first ...

  20. Evaluation of insulin medium or chondrogenic medium on proliferation and chondrogenesis of ATDC5 cells.

    Science.gov (United States)

    Yao, Yongchang; Zhai, Zhichen; Wang, Yingjun

    2014-01-01

    The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

  1. Evaluation of Insulin Medium or Chondrogenic Medium on Proliferation and Chondrogenesis of ATDC5 Cells

    Directory of Open Access Journals (Sweden)

    Yongchang Yao

    2014-01-01

    Full Text Available Background. The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM was used to induce chondrogenesis while chondrogenic medium (CM, which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs, was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM on chondrogenesis of ATDC5 cells. Methods. ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. Results. Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. Conclusion. This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

  2. Architecture of Institution & Home. Architecture as Cultural Medium

    NARCIS (Netherlands)

    Robinson, J.W.

    2004-01-01

    This dissertation addresses how architecture functions as a cultural medium. It does so by by investigating how the architecture of institution and home each construct and support different cultural practices. By studying the design of ordinary settings in terms of how qualitative differences in

  3. 21 CFR 866.2390 - Transport culture medium.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Transport culture medium. 866.2390 Section 866.2390 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2390 Transport culture...

  4. Effect of different surfactants in biorelevant medium on the secretion of a lipophilic compound in lipoproteins using Caco-2 cell culture

    DEFF Research Database (Denmark)

    Karpf, Ditte M; Holm, René; Garafalo, Carole

    2006-01-01

    The impact of a pharmaceutical relevant metabolizable, ionic surfactant or two synthetic, nonionic surfactants on the absorption and lipoprotein incorporation of a lipophilic drug, retinol, was studied in the Caco-2 cell culture. Filter-grown monolayers of Caco-2 cells were incubated for 20 h...... was ultracentrifugated into different lipoprotein classes and their (3)H-retinol and (14)C-lipid concentrations were determined. The cells incubated with lyso-PC and Tween 80 increased the incorporation of (3)H-retinol and (14)C-lipid into chylomicrons and very low density lipoproteins (VLDL). The explored surfactants...... impacted the incorporation of (3)H-retinol and (14)C-lipid in chylomicrons and VLDL in a concentration-dependent manner. As these surfactants interfere with the intestinal lipoprotein secretion, inclusion of high concentrations of the surfactants in lipid-based formulations of poorly aqueous soluble drugs...

  5. Aquaculture biological waste as culture medium to cultivation of Ankistrodesmus gracilis (Reinsch) Korshikov.

    Science.gov (United States)

    Sipaúba-Tavares, L H; Florêncio, T; Scardoeli-Truzzi, B

    2017-11-21

    Current study investigated the effectiveness of different macrophytes as culture media for Ankistrodesmus gracilis in laboratory conditions. Significant difference (p medium (CHU12) and macrophytes culture media. Mean cell density in NPK, Eichhornia crassipes and E. azurea media was higher (p medium. Chlorophyll-a was higher than 1 g.L-1, except in CHU12 (0.7 ± 0.4 g.L-1) and T. domingensis (0.8 ± 0.3 g.L-1) media. Nitrate decreased sharply as from the 7th-day of the experiment. Ammonium and total phosphorus were highest in culture media and ranged between 0.4 g.L-1 (P. cordata medium) and 1.7 g.L-1 (CHU12 medium) for ammonium, and between 0.8 g.L-1 (CHU12 medium) and 1.9 g.L-1 (T. domingensis medium) for total phosphorus. Results revealed inorganic fertilizer and macrophytes combined with vitamins may be effective as culture media and strongly supports the growth of Ankistrodesmus gracilis, since cell density and biochemical composition are similar to or higher than conventional culture medium (CHU12). Macrophyte is a tool for aquaculture since biological wastes may be used with nutrients to improve the cultivation of microalgae.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  7. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled c...... compared to cell cultured in culture flasks incubated in a dark and CO2 conditioned incubator....

  8. Optimization of ferric chloride concentration and pH to improve both cell growth and flocculation in Chlorella vulgaris cultures. Application to medium reuse in an integrated continuous culture bioprocess.

    Science.gov (United States)

    Lecina, Martí; Nadal, Gisela; Solà, Carles; Prat, Jordi; Cairó, Jordi J

    2016-09-01

    Combined effect of ferric chloride and pH on Chlorella vulgaris growth and flocculation were optimized using DoE. Afterwards, an integrated bioprocess for microalgae cultivation and harvesting conceived as a sole step was run in continuous operation mode. Microalgae concentration in a 2L-photobioreactor was about 0.5gL(-1) and the efficiency of flocculation in the coupled sedimentation tank was about 95%. Dewatered microalgae reached a biomass concentrations increase about 50-fold, whereas it was only about 0.02gL(-1) in the clarified medium. Then, the reuse of the clarified medium recovered was further evaluated. The clarified medium was reused without any further nutrient supplementation, whereas a second round of medium reuse was performed after supplementation of main nutrients (phosphate-sulfate-nitrate), micronutrients and ferric chloride. The medium reuse strategy did not affect cell growth and flocculation. Consequently, the reuse of medium reduces the nutrients requirements and the demand for water, and therefore the production costs should be reduced accordingly. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  10. Optimum culture medium composition for lipopeptide production by ...

    Indian Academy of Sciences (India)

    Optimum culture medium composition for lipopeptide production by Bacillus subtilis using response surface model-based ant colony optimization. J SATYA ESWARI1, M ANAND2,∗ and C VENKATESWARLU1. 1Chemical Engineering Sciences Division, Indian Institute of Chemical Technology,. Hyderabad 500007, India.

  11. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

    Science.gov (United States)

    Oliveira-Rodríguez, Myriam; López-Cobo, Sheila; Reyburn, Hugh T; Costa-García, Agustín; López-Martín, Soraya; Yáñez-Mó, María; Cernuda-Morollón, Eva; Paschen, Annette; Valés-Gómez, Mar; Blanco-López, Maria Carmen

    2016-01-01

    Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

  12. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

    Directory of Open Access Journals (Sweden)

    Myriam Oliveira-Rodríguez

    2016-08-01

    Full Text Available Exosomes are cell-secreted nanovesicles (40–200 nm that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

  13. [Evaluation of chromogenic medium Uriselect4 in urine culture].

    Science.gov (United States)

    Ferjani, Asma; Marzouk, Manel; Idriss, Nadia; Sammoud, Sammoud; Hannachi, Naila; Boukadida, Janel

    2011-01-01

    We evaluated the performance and the cost of chromogenic medium Uriselect4 agar with regard to the standard medium for the detection and identification of urinary tract pathogens. A total of 503 clinical urine specimens containing leucocytes greater or equal to 104/mL were analysed prospectively, in parallel by two different persons on blood agar (GS) and Uriselect4 according to the manufacturers' instructions. Of the 503 urine specimens tested, 210 gave a positive culture on Uriselect4 versus 181 on GS. The majority of bacterial species grew on both media; enterobacteria grew on Uriselect4 better than GS. The identification of Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), KES group and Enterococcus faecalis (E. faecalis) did not require the use of galleries Api and has a gain of 24  h. Positive pure cultures on Uriselect4 corresponding to negative cultures of GS were noted in 17 ases. Conversely, in seven cases a positive pure culture on GS was noted while the corresponding Uriselect4 cultures were negative. The cost of identification on GS (including the cost of galleries Api), was about two times higher than Uriselect4. Uriselect4 medium isolates the most frequent urinary tract pathogens and identify them so almost immediately, with a lower cost.

  14. Sugarcane Bagasse: A Potential Medium for Fungal Cultures

    Directory of Open Access Journals (Sweden)

    Arushdeep Sidana

    2014-01-01

    Full Text Available Worldwide, sugarcane industries produce tons of sugarcane bagasse as residual/waste material. This residual material is rich in complex lignocellulosic substances and may be used as a low cost carbon and energy source for the growth of fungal species. The present work was aimed at designing a sugarcane waste-based medium as a substitute for expensive commercial media for growing fungal cultures. Eight species of fungi, namely, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Fusarium sp., and four unidentified species F1, F2, F3, and F5, were grown on the sugarcane bagasse medium which produced remarkable results and competed with standard media like potato dextrose agar, Sabouraud dextrose agar, and cornmeal agar. The designed medium was able to provide nourishment to the fungi as well as prevent the growth of any bacterial or fungal contaminant. The production of spores was more in the sugarcane medium as compared with standard media. Hence, this study led to the discovery of a new and efficient medium for fungal cultures as well as decrease in the waste disposal expenses and efforts.

  15. A defined growth medium with very low background carbon for culturing Clostridium thermocellum.

    Science.gov (United States)

    Holwerda, Evert K; Hirst, Kyle D; Lynd, Lee R

    2012-06-01

    A growth medium was developed for cultivation of Clostridium thermocellum ATCC 27405 in which "background" carbon present in buffers, reducing agents, chelating agents, and growth factors was a small fraction of the carbon present in the primary growth substrate. Background carbon was 1.6% of primary substrate carbon in the low-carbon (LC) medium, whereas it accounts for at least 40% in previously reported media. Fermentation of cellulose in LC medium was quite similar to Medium for Thermophilic Clostridia (MTC), a commonly used growth medium that contains background carbon at 88% of primary substrate carbon. Of particular note, we found that the organism can readily be cultivated by eliminating some components, lowering the concentrations of others, and employing a tenfold lower concentration of reducing agent. As such, we were able to reduce the amount of background carbon 55-fold compared to MTC medium while reaching the same cell biomass concentration. The final mass ratios of the products acetate:ethanol:formate were 5:3.9:1 for MTC and 4.1:1.5:1 for LC medium. LC medium is expected to facilitate metabolic studies involving identification and quantification of extracellular metabolites. In addition, this medium is expected to be useful in studies of cellulose utilization by anaerobic enrichment cultures obtained from environmental inocula, and in particular to diminish complications arising from metabolism of carbon-containing compounds other than cellulose. Finally, LC medium provides a starting point for industrial growth media development.

  16. Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

    Science.gov (United States)

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

    1999-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

  17. Aleuria aurantia - indole metabolites of fruit bodies, mycelial culture and culture medium

    Directory of Open Access Journals (Sweden)

    Janina Węgiel

    2014-08-01

    Full Text Available The aim of present study was to investigate and compare indole metabolites of fruit bodies, mycelium cultivated in vitro and culture medium of the fungus Aleuria aurantia (Fr. Fuck. By use of a number of chromatographic and spectroscopic methods several indole metabolites have been detected and identified among other the 3-indolebutyric acid was produced and extracted to the culture medium. Furthermore 3-indoleatonitrile and tryptophane degradative products have been found both in fruit bodies and mycelium.

  18. Growth and carotenogenesis in Rhodosporidium diobovatum IMB Y-5023: effects of culture medium and illumination intensity.

    Science.gov (United States)

    Ielchishcheva, Iuliia; Stachowiak, Barbara; Szwengiel, Artur; Bozhkov, Anatoliy

    2018-01-01

    The oleaginous yeast Rhodosporidium diobovatum is a poorly described producer of carotenoids and may be of interest in biotechnology. This study investigated the effects of culture medium and illumination on effective carotenoid production by R. diobovatum IMB Y-5023. Yeast was cultured on carrot, bran extract, and modified yeast malt (YM) medium at illuminations in the 0-5000 lx range. Biomass, total carotenoids and their profile were assessed after cultivation. In order to compare samples, cluster analysis and principal component analysis were used to visualize the relationships between the variables and samples. Results show that it is not illumination but culture medium that is the main factor determining the quantity and proportions of carotenoids produced by R. diobovatum IMB Y-5023. The yeast grew and produced pigments throughout the tested range of illumination intensity. The modified YM medium was optimal for carotenogenesis. In cultures on that medium, the highest carotenoid yields of 24.98 mg g-1 dry cell weight and 77 mg L-1 were recorded. It was found that this yeast is capable of assimilating oligosaccharides and can grow and produce carotenoids in low-glucose media containing DP3 and DP4. Moreover R. diobovatum IMB Y-5023 produced lycopene as the main pigment independently of the culture conditions. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Basic cell culture.

    Science.gov (United States)

    Pollard, J W

    1990-01-01

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

  20. Evaluating in Vitro Culture Medium of Gut Microbiome with Orthogonal Experimental Design and a Metaproteomics Approach.

    Science.gov (United States)

    Li, Leyuan; Zhang, Xu; Ning, Zhibin; Mayne, Janice; Moore, Jasmine I; Butcher, James; Chiang, Cheng-Kang; Mack, David; Stintzi, Alain; Figeys, Daniel

    2018-01-05

    In vitro culture based approaches are time- and cost-effective solutions for rapidly evaluating the effects of drugs or natural compounds against microbiomes. The nutritional composition of the culture medium is an important determinant for effectively maintaining the gut microbiome in vitro. This study combines orthogonal experimental design and a metaproteomics approach to obtaining functional insights into the effects of different medium components on the microbiome. Our results show that the metaproteomic profile respond differently to medium components, including inorganic salts, bile salts, mucin, and short-chain fatty acids. Multifactor analysis of variance further revealed significant main and interaction effects of inorganic salts, bile salts, and mucin on the different functional groups of gut microbial proteins. While a broad regulating effect was observed on basic metabolic pathways, different medium components also showed significant modulations on cell wall, membrane, and envelope biogenesis and cell motility related functions. In particular, flagellar assembly related proteins were significantly responsive to the presence of mucin. This study provides information on the functional influences of medium components on the in vitro growth of microbiome communities and gives insight on the key components that must be considered when selecting and optimizing media for culturing ex vivo microbiotas.

  1. Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition

    OpenAIRE

    Liu, Jing; Saponjian, Yero; Mahoney, Mark M.; Staley, Kevin J.; Berdichevsky, Yevgeny

    2017-01-01

    Rodent organotypic hippocampal cultures spontaneously develop epileptiform activity after approximately 2 weeks in vitro and are increasingly used as a model of chronic post-traumatic epilepsy. However, organotypic cultures are maintained in an artificial environment (culture medium), which contains electrolytes, glucose, amino acids and other components that are not present at the same concentrations in cerebrospinal fluid (CSF). Therefore, it is possible that epileptogenesis in organotypic ...

  2. New insights into the effect of medium-chain-length lactones on yeast membranes. Importance of the culture medium.

    Science.gov (United States)

    Ta, Thi Minh Ngoc; Cao-Hoang, Lan; Phan-Thi, Hanh; Tran, Hai Dang; Souffou, Nadhuirata; Gresti, Joseph; Marechal, Pierre-André; Cavin, Jean-François; Waché, Yves

    2010-07-01

    In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to gamma-dodecalactone. After a 60-min exposure to 3 g L(-1), about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L(-1) did not alter cultivability despite some transient loss of membrane integrity from 3 g L(-1). It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-beta-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.

  3. The species origin of the serum in the culture medium influences the in vitro toxicity of silica nanoparticles to HepG2 cells.

    Science.gov (United States)

    Pisani, Cédric; Rascol, Estelle; Dorandeu, Christophe; Gaillard, Jean-Charles; Charnay, Clarence; Guari, Yannick; Chopineau, Joël; Armengaud, Jean; Devoisselle, Jean-Marie; Prat, Odette

    2017-01-01

    The formation of a protein corona around nanoparticles can influence their toxicity, triggering cellular responses that may be totally different from those elicited by pristine nanoparticles. The main objective of this study was to investigate whether the species origin of the serum proteins forming the corona influences the in vitro toxicity assessment of silica nanoparticles. Coronas were preformed around nanoparticles before cell exposures by incubation in fetal bovine (FBS) or human (HS) serum. The compositions of these protein coronas were assessed by nano-LC MS/MS. The effects of these protein-coated nanoparticles on HepG2 cells were monitored using real-time cell impedance technology. The nanoparticle coronas formed in human or fetal bovine serum comprised many homologous proteins. Using human compared with fetal bovine serum, nanoparticle toxicity in HepG2 cells decreased by 4-fold and 1.5-fold, when used at 50 and 10μg/mL, respectively. It is likely that "markers of self" are present in the serum and are recognized by human cell receptors. Preforming a corona with human serum seems to be more appropriate for in vitro toxicity testing of potential nanocarriers using human cells. In vitro cytotoxicity assays must reflect in vivo conditions as closely as possible to provide solid and useful results.

  4. 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.

  5. 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

  6. Pectinase Production in a Defined Medium Using Surface Culture Fermentation

    Directory of Open Access Journals (Sweden)

    Haidar Abbasi

    2010-10-01

    Full Text Available Two pectinase producing fungi species were isolated. Using a defined mineral medium and pectin as the carbon source, the capability of the species to produce pecinase was investigated in surface culture fermentation. The results showed that Aspergillus niger performs better than Thericoderma reesei in term of pectinase production, glucose has a repressive effect on pectinase production, and among nitrogen sources of ammonium sulfate, yeast extract , and sodium nitrate, ammonium sulfate is the best. The maximum exo-pectinase obtained in this research was about 1.7U/mL and the maximum endo-pectinase activity was about 0.015U/mL. Pectinase production was very weak when pectin was substituted by sugar beet pulp probably due to inefficient contact between the microorganism and substrate particles in surface culture fermentation. Using sugar beet pulp in solid-state fermentation gave results comparable with those obtained in surface culture fermentation of pectin and in this research was investigated the feasibility of continuous surface culture fermentation for pectinases production. The bioreactor was placed in an incubator at 35ºC.

  7. Dependence of the cytotoxicity of multi-walled carbon nanotubes on the culture medium

    Science.gov (United States)

    Zhu, Ying; Ran, Tiecheng; Li, Yuguo; Guo, Jinxue; Li, Wenxin

    2006-09-01

    This study examined the influence of multi-walled carbon nanotubes (MWNTs) on the growth of the unicellular protozoan Tetrahymena pyriformis. Contrary to the findings from most other investigations, our experiment indicated that MWNTs stimulated growth of the cells cultured in proteose peptone yeast extract medium (PPY). Atomic force microscopy images and thermogravimetric analysis showed the spontaneous formation of peptone-MWNT conjugates in the medium by noncovalent binding. Uptake of large amounts of the conjugates by Tetrahymena pyriformis was responsible for growth stimulation, evidenced by images with fluorescently labelled peptone. After the PPY medium was replaced by a filtrated pond water medium (FPW), however, inhibition of the growth of cells exposed to MWNTs occurred. Measurements of the level of malondialdehyde and superoxide dismutase activity demonstrated further that MWNTs might be either toxic or nontoxic, depending on the medium used to cultivate Tetrahymena pyriformis. The biological effects of the interaction of MWNTs with some composites in culture media would be helpful for understanding the mechanisms of the toxicity of carbon nanotubes to living systems.

  8. Dependence of the cytotoxicity of multi-walled carbon nanotubes on the culture medium

    International Nuclear Information System (INIS)

    Zhu Ying; Ran Tiecheng; Li Yuguo; Guo Jinxue; Li Wenxin

    2006-01-01

    This study examined the influence of multi-walled carbon nanotubes (MWNTs) on the growth of the unicellular protozoan Tetrahymena pyriformis. Contrary to the findings from most other investigations, our experiment indicated that MWNTs stimulated growth of the cells cultured in proteose peptone yeast extract medium (PPY). Atomic force microscopy images and thermogravimetric analysis showed the spontaneous formation of peptone-MWNT conjugates in the medium by noncovalent binding. Uptake of large amounts of the conjugates by Tetrahymena pyriformis was responsible for growth stimulation, evidenced by images with fluorescently labelled peptone. After the PPY medium was replaced by a filtrated pond water medium (FPW), however, inhibition of the growth of cells exposed to MWNTs occurred. Measurements of the level of malondialdehyde and superoxide dismutase activity demonstrated further that MWNTs might be either toxic or nontoxic, depending on the medium used to cultivate Tetrahymena pyriformis. The biological effects of the interaction of MWNTs with some composites in culture media would be helpful for understanding the mechanisms of the toxicity of carbon nanotubes to living systems

  9. Lactated Ringer-based storage solutions are equally well suited for the storage of fresh osteochondral allografts as cell culture medium-based storage solutions.

    Science.gov (United States)

    Harb, Afif; von Horn, Alexander; Gocalek, Kornelia; Schäck, Luisa Marilena; Clausen, Jan; Krettek, Christian; Noack, Sandra; Neunaber, Claudia

    2017-07-01

    Due to the rising interest in Europe to treat large cartilage defects with osteochondrale allografts, research aims to find a suitable solution for long-term storage of osteochondral allografts. This is further encouraged by the fact that legal restrictions currently limit the use of the ingredients from animal or human sources that are being used in other regions of the world (e.g. in the USA). Therefore, the aim of this study was A) to analyze if a Lactated Ringer (LR) based solution is as efficient as a Dulbecco modified Eagle's minimal essential medium (DMEM) in maintaining chondrocyte viability and B) at which storage temperature (4°C vs. 37°C) chondrocyte survival of the osteochondral allograft is optimally sustained. 300 cartilage grafts were collected from knees of ten one year-old Black Head German Sheep. The grafts were stored in four different storage solutions (one of them DMEM-based, the other three based on Lactated Ringer Solution), at two different temperatures (4 and 37°C) for 14 and 56days. At both points in time, chondrocyte survival as well as death rate, Glycosaminoglycan (GAG) content, and Hydroxyproline (HP) concentration were measured and compared between the grafts stored in the different solutions and at the different temperatures. Independent of the storage solutions tested, chondrocyte survival rates were higher when stored at 4°C compared to storage at 37°C both after short-term (14days) and long-term storage (56days). At no point in time did the DMEM-based solution show a superior chondrocyte survival compared to lactated Ringer based solution. GAG and HP content were comparable across all time points, temperatures and solutions. LR based solutions that contain only substances that are approved in Germany may be just as efficient for storing grafts as the USA DMEM-based solution gold standard. Moreover, in the present experiment storage of osteochondral allografts at 4°C was superior to storage at 37°C. Copyright © 2017

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

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

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

  11. Responses of NBT-II bladder carcinoma cells to conditioned medium from normal fetal urogenital sinus.

    Science.gov (United States)

    Rowley, D R; Tindall, D J

    1987-06-01

    In vitro studies were conducted to determine whether conditioned medium from rat fetal urogenital sinus explants would affect phenotypic characteristics of NBT-II urinary bladder carcinoma cells in culture. NBT-II cells were exposed to medium (30%, v/v) conditioned for 48 h by intact urogenital sinus explants derived from 18-day fetal rats. Upon exposure for 23 h the [3H]thymidine incorporation by NBT-II cells was decreased by 40.3% relative to control cultures. This effect was paralleled by a similar decrease in proliferation. NBT-II cultures decreased in cell number by 32.1 and 45.8% on days 2 and 4, respectively, after exposure to conditioned medium. Although cell proliferation was inhibited, conditioned medium acted to induce an increase in protein secretion. An increase of 18.6% was observed in the incorporation of [35S]methionine into newly synthesized, secreted proteins by NBT-II cells exposed to conditioned medium for 23 h. Morphologically the NBT-II cells exposed to conditioned medium were larger, more spread out, and exhibited a greater array of lamellipodia and filopodia, although [35S]methionine incorporation into cellular proteins was decreased by 11.1%. These results suggest that diffusable factors produced by fetal urogenital sinus explants can induce changes in proliferation, protein synthesis, protein secretion, and phenotypic morphology of NBT-II carcinoma cells in culture.

  12. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

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

  13. 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.

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

    International Nuclear Information System (INIS)

    Felker, F.C.

    1990-01-01

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

  15. Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

    Directory of Open Access Journals (Sweden)

    Hui Sun

    Full Text Available Administration of mesenchymal stromal cells (MSC improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice, NSC-34 cells and glial cells (astrocytes, microglia (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM attenuated staurosporine (STS - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF and ciliary neurotrophic factor (CNTF gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1 was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

  16. Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele

    2011-07-01

    For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

  17. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

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

  18. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    Science.gov (United States)

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

  19. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    Science.gov (United States)

    Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Trinh, Tinh T. (Inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  20. Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

    Science.gov (United States)

    Alves da Silva, M L; Costa-Pinto, A R; Martins, A; Correlo, V M; Sol, P; Bhattacharya, M; Faria, S; Reis, R L; Neves, Nuno M

    2015-06-01

    Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Somatic embryogenesis on Musa AAAB, cv. FHIA-18, using liquids culture mediums

    Directory of Open Access Journals (Sweden)

    Luis A. Barranco

    2002-04-01

    Full Text Available Homogenous cell suspensions were iniciated from somatic embryos in the globular stage and the greatest volume of cell biomass on multiplying the suspensions at a density of 3.0% PCV. From the fifteenth day in culture medium for the formation of embryos, structures consisting of proembryos and somatic embryos in the globular stage started to form. With respect to the densities studied, the best results were obtained with 100 mgFW, where 1 871 SE.l-1 formed with a weight of 248 mgFW.l-1 after 30 days. With an initial density of 0.6 gFW in the culture medium for secondary multiplication, an increase of 42.9-fold the initial amount of fresh weight was obtained; after 60 days of culture 15 985 SE.l-1 were obtained. The greatest percentage of maturation was obtained with 400 mgFW with 70% of mature somatic embryos. The positive effect of Biobras-6 (brassinosteroid analogous was confirmed, with a concentration of 0.01 mg.l-1 the best germination percentages were obtained in liquid and semisolid culture medium. Embryo germination in temporaly inmersion (RITA was achieved with an inoculum density of 0.5 gFW for system with 85% germination. One thousand plants obtained from somatic embryos were taken to ex vitro environment, along with plants derived from conventional micropropagation (shoot tips to carry out studies on the possible presence of somaclonal variation. During the first cycle of production, the plants derived from the two methods in vitro culture showed differences with respect to the plants derived from corms in height, diameter and number of suckers. In the second production cycle, the plants from somatic embryos showed similar characteristics to the plants derived from shoot tip and corms with respect to the morphological parameters evaluated, with only 0.2% of the plants with phenotypic changes. Key Words: Banana, cellular density, germination, somaclonal variability, somatic embryo

  2. Does supplementation of in-vitro culture medium with melatonin improve IVF outcome in PCOS?

    Science.gov (United States)

    Kim, Mi Kyoung; Park, Eun A; Kim, Hyung Joon; Choi, Won Yun; Cho, Jung Hyun; Lee, Woo Sik; Cha, Kwang Yul; Kim, You Shin; Lee, Dong Ryul; Yoon, Tae Ki

    2013-01-01

    Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (PPregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer. Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the

  3. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  4. Effect of Nitrogen Sources in Culture Medium on L-Lactate Fermentation Employing Lactococcus Zactis IO-1

    OpenAIRE

    Tanaka, Kenji; Ohta, Tomomi; Ishizaki, Ayaaki; 田中, 賢二; 太田, 智美; 石崎, 文彬

    1995-01-01

    Effect of amino acids, yeast extract and polypeptone on cell growth and L-lactate production of Lactococcus laclis IO-l was investigated. The growth rate of the microorganism, cultivated in a synthetic medium consisting of amino acid, vitamins and minerals, is lower than that of the media containing yeast extract. It was considered that yeast extract was essential for rapid growth of L. lactis IO-1. An increase in yeast extract concentration in the culture medium increased specific growth rat...

  5. Conditioned Medium from Periodontal Ligament Stem Cells Enhances Periodontal Regeneration.

    Science.gov (United States)

    Nagata, Mizuki; Iwasaki, Kengo; Akazawa, Keiko; Komaki, Motohiro; Yokoyama, Naoki; Izumi, Yuichi; Morita, Ikuo

    2017-05-01

    Periodontal disease is one of the most common infectious diseases in adults and is characterized by the destruction of tooth-supporting tissues. Mesenchymal stem cells (MSCs) comprise the mesoderm-originating stem cell population, which has been studied and used for cell therapy. However, because of the lower rate of cell survival after MSC transplantation in various disease models, paracrine functions of MSCs have been receiving increased attention as a regenerative mechanism. The aim of this study was to investigate the regenerative potential of transplanted conditioned medium (CM) obtained from cultured periodontal ligament stem cells (PDLSCs), the adult stem cell population in tooth-supporting tissues, using a rat periodontal defect model. Cell-free CM was collected from PDLSCs and fibroblasts, using ultrafiltration and transplanted into surgically created periodontal defects. Protein content of CM was examined by antibody arrays. Formation of new periodontal tissues was analyzed using microcomputed tomography and histological sections. PDLSC-CM transplantation enhanced periodontal tissue regeneration in a concentration-dependent manner, whereas fibroblast-CM did not show any regenerative function. Proteomic analysis revealed that extracellular matrix proteins, enzymes, angiogenic factors, growth factors and cytokines were contained in PDLSC-CM. Furthermore, PDLSC-CM transplantation resulted in the decreased mRNA level of tumor necrosis factor-α (TNF-α) in healing periodontal tissues. In addition, we found that PDLSC-CM suppressed the mRNA level of TNF-α in the monocyte/macrophage cell line, RAW cells, stimulated with IFN-γ. Our findings suggested that PDLSC-CM enhanced periodontal regeneration by suppressing the inflammatory response through TNF-α production, and transplantation of PDLSC-CM could be a novel approach for periodontal regenerative therapy.

  6. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...

  7. Effect of genotypes and culture medium on shoot regeneration and ...

    African Journals Online (AJOL)

    For proliferation, regenerated shoots in TDZ were subcultured in medium supplemented with 0.2, 0.4 and 0.6 mg/l TDZ, 2 mg/l BA or 2 mg/l Kin and regenerated shoots in BA were subcultured on the induction medium. In the second phase, mediums of MS, 1/2 MS, MS with 1/2 NH4NO3 and KNO3 concentration (MS-1/2N), ...

  8. Modification of MCF-10A Cells with Pioglitazone and Serum-Rich Growth Medium Increases Soluble Factors in the Conditioned Medium, Likely Reducing BT-474 Cell Growth

    Directory of Open Access Journals (Sweden)

    Kalpanah Nadarajan

    2012-05-01

    Full Text Available In the present study, we aimed to preincubate MCF-10A cells with pioglitazone and/or serum-rich growth media and to determine adhesive and non-adhesive interactions of the preincubated MCF-10A cells with BT-474 cells. For this purpose, the MCF-10A cells were preincubated with pioglitazone and/or serum-rich growth media, at appropriate concentrations, for 1 week. The MCF-10A cells preincubated with pioglitazone and/or serum-rich growth media were then co-cultured adhesively and non-adhesively with BT-474 cells for another week. Co-culture of BT-474 cells with the preincubated MCF-10A cells, both adhesively and non-adhesively, reduced the growth of the cancer cells. The inhibitory effect of the preincubated MCF-10A cells against the growth of BT-474 cells was likely produced by increasing levels of soluble factors secreted by the preincubated MCF-10A cells into the conditioned medium, as immunoassayed by ELISA. However, only an elevated level of a soluble factor distinguished the conditioned medium collected from the MCF-10A cells preincubated with pioglitazone and serum-rich growth medium than that with pioglitazone alone. This finding was further confirmed by the induction of the soluble factor transcript expression in the preincubated MCF-10A cells, as determined using real-time PCR, for the above phenomenon. Furthermore, modification of the MCF-10A cells through preincubation did not change the morphology of the cells, indicating that the preincubated cells may potentially be injected into mammary fat pads to reduce cancer growth in patients or to be used for others cell-mediated therapy.

  9. Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

    Directory of Open Access Journals (Sweden)

    Lucas Felipe de Oliveira

    2015-01-01

    Full Text Available Systemic arterial hypertension (SAH, a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs, which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2 increases their therapeutic effects in spontaneously hypertensive rats (SHRs. Adult female SHRs were administered an intraperitoneal injection of vehicle solution n=10, MSCs cultured in conventional medium (DMEM plus 10% FBS, n=11, or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n=10. Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH.

  10. Development and validation of a minimal growth medium for recycling Chlorella vulgaris culture.

    Science.gov (United States)

    Hadj-Romdhane, F; Jaouen, P; Pruvost, J; Grizeau, D; Van Vooren, G; Bourseau, P

    2012-11-01

    When microalgae culture medium is recycled, ions (e.g. Na(+), K(+), Ca(2+)) that were not assimilated by the microalgae accumulate in the medium. Therefore, a growth medium (HAMGM) was developed that included ions that were more easily assimilated by Chlorella vulgaris, such as ammonium one (NH(4)(+)). Recycling performance was studied by carrying out 8-week continuous cultivation of C. vulgaris with recycled HAMGM medium. No loss of biomass productivity was observed compared to culture in a conventional medium, and accumulation of ions over time was negligible. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. IN-VITRO CULTURE FOR REGENERATION OF Melia azedarach L. USING AGITATED LIQUID MEDIUM

    OpenAIRE

    Nirsatmanto Arif

    2006-01-01

    This study investigated the applicability of liquid medium in direct organogenesis of in-vitro culture of Melia azedarach L. Explants were collected from in-vitro multiplication originating from aseptically germinated seedling and from a 48 - years old mature tree. For adventitious shoot differentiation, 2 mm length of excised explants were cultured on liquid medium of MS (Murashige and Skoog's) basal medium supplemented with 28 combinations of hormone BAP (6-benzylaminopurine) and NAA (á- na...

  12. Optimization of Culture Medium Enhances Viable Biomass Production and Biocontrol Efficacy of the Antagonistic Yeast, Candida diversa

    Directory of Open Access Journals (Sweden)

    Jia Liu

    2017-10-01

    Full Text Available Viable biomass production is a key determinant of suitability of antagonistic yeasts as potential biocontrol agents. This study investigated the effects of three metal ions (magnesium, ferrous, and zinc on biomass production and viability of the antagonistic yeast, Candida diversa. Using response surface methodology to optimize medium components, a maximum biomass was obtained, when the collective Mg2+, Fe2+, and Zn2+ concentrations were adjusted in a minimal mineral (MM medium. Compared with the unmodified MM, and three ion-deficient MM media, yeast cells cultured in the three ion-modified MM medium exhibited a lower level of cellular oxidative damage, and a higher level of antioxidant enzyme activity. A biocontrol assay indicated that C. diversa grown in the ion-modified MM exhibited the greatest level of control of gray mold on apple fruit. These results provide new information on culture medium optimization to grow yeast antagonists in order to improve biomass production and biocontrol efficacy.

  13. Development of detection medium for hard-to-culture beer-spoilage lactic acid bacteria.

    Science.gov (United States)

    Suzuki, K; Asano, S; Iijima, K; Kuriyama, H; Kitagawa, Y

    2008-05-01

    To develop a detection medium for hard-to-culture beer-spoilage lactic acid bacteria (LAB). Four hard-to-culture beer-spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild-type strains in beer. To develop a countermeasure against these hard-to-culture beer-spoilage LAB, a beer-based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard-to-culture beer-spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer-based medium. Further comparative study was performed with five other media widely used for the detection of beer-spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer-spoiler detection (ABD) medium, possessed superior sensitivity for hard-to-culture beer-spoilage LAB and comparable sensitivity with easy-to-culture beer-spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro-organisms, and thereby allow the selective growth of beer-spoilage LAB. Advanced beer-spoiler detection medium is considered as an effective tool for comprehensive detection of beer-spoilage LAB in breweries. The detection by ABD medium can be used as an indicator for differentiating the beer-spoilage ability of LAB without further confirmatory tests in breweries.

  14. Maintenance and neuronal cell differentiation of neural stem cells C17.2 correlated to medium availability sets design criteria in microfluidic systems.

    Science.gov (United States)

    Wang, Bu; Jedlicka, Sabrina; Cheng, Xuanhong

    2014-01-01

    Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes. A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10(4) µm3/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10(4) µm3/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length. MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in

  15. The effect of medium composition on ovary-slice culture and ovule culture in intraspecific Tulipa gesneriana crosses

    NARCIS (Netherlands)

    Creij, van M.G.M.; Kerckhoffs, D.M.F.J.; Bruijn, de S.M.; Tuyl, van J.M.; Vreugdenhil, D.

    2000-01-01

    The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for

  16. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  17. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

    OpenAIRE

    Chieh-Lun Liu; Aaron M. Watson; Allen R. Place; Rosemary Jagus

    2017-01-01

    Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gr...

  18. RSW Fully Tet Medium Cell-Centered Mesh

    Data.gov (United States)

    National Aeronautics and Space Administration — This is the RSW Fully tetrahedral medium cell-centered unstructured grid with a viscous wall. UG3 : Grid File Name = rsw_med_tetcc.b8.ugrid UG3 : Quad Surface Faces=...

  19. RSW Mixed Element Cell-Centered Medium Mesh

    Data.gov (United States)

    National Aeronautics and Space Administration — This RSW gridset is designed as the medium size mixed element grid for use with cell-centered unstructured meshes. UG3 : Grid File Name = rsw_med_mixedcc.b8.ugrid...

  20. Preirradiation of medium induces a subsequent stimulation or inhibition of growth according to the physiological state in Synechococcus lividus in culture

    International Nuclear Information System (INIS)

    Conter, A.

    1987-01-01

    The proliferation of Synechococcus lividus cells grown in preirradiated medium was compared with the proliferation of cells grown in a shielded or freshly prepared medium. Aging of medium in a shielded chamber resulted in a slight inhibiting effect on growth in every phase of the cell cycle which was used. Preirradiation of medium resulted in a stimulation of growth observed on Day 7 in cultures inoculated with cells selected in the deceleration phase and an inhibition of growth in cultures inoculated with exponentially growing cells. Addition of catalase (100 U X ml-1) counteracted the stimulating effect but did not modify the inhibiting effect induced by preirradiated medium. Results demonstrated the indirect effect of low doses of irradiation, implying the presence of hydrogen peroxide in radiostimulation and other radioproducts in the inhibitory effect

  1. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  2. 21 CFR 866.2320 - Differential culture medium.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2320 Differential culture... biological materials intended for medical purposes to cultivate and identify different types of pathogenic...

  3. 21 CFR 866.2300 - Multipurpose culture medium.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2300 Multipurpose culture... or solid biological materials intended for medical purposes for the cultivation and identification of...

  4. 21 CFR 866.2360 - Selective culture medium.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2360 Selective culture... solid biological materials intended for medical purposes to cultivate and identify certain pathogenic...

  5. 21 CFR 866.2330 - Enriched culture medium.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture... solid biological materials intended for medical purposes to cultivate and identify fastidious...

  6. Phenotypic diversity of patient-derived melanoma populations in stem cell medium.

    Science.gov (United States)

    Sztiller-Sikorska, Malgorzata; Hartman, Mariusz L; Talar, Beata; Jakubowska, Justyna; Zalesna, Izabela; Czyz, Malgorzata

    2015-06-01

    Melanomas are highly heterogeneous tumors and there is no treatment effective at achieving long-term remission for metastatic melanoma patients. Thus, an appropriate model system for studying melanoma biology and response to drugs is necessary. It has been shown that composition of the medium is a critical factor in preserving the complexity of the tumor in in vitro settings, and melanospheres maintained in stem cell medium are a good model in this respect. In the present study, we observed that not all nodular melanoma patient-derived cell populations grown in stem cell medium were capable of forming melanospheres, and cell aggregates and anchorage-independent single-cell cultures emerged instead. Self-renewing capacity and unlimited growth potential indicated the presence of cells with stem-like properties in all patient-derived populations but immunophenotype and MITF expression exhibited variability. Enhanced MITF expression and activity was observed in melanospheres in comparison with cell aggregates and single-cell culture, and hypoxic-like conditions that increased the ability of single-cell population to form melanospheres enhanced MITF expression and cell pigmentation as well. Thus, MITF seems to be a critical transcription factor for formation of both patient-derived and hypoxia-induced melanospheres. After 2 years of continuous culturing, melanospheres progressively underwent transition into cell aggregates that was accompanied by changes in expression of several MITF-dependent genes associated with melanogenesis and survival and alterations in the composition of subpopulations but not in the frequency of ABCB5-positive cells. Several biological properties of parent tumor are well preserved in patient-derived melanospheres, but during prolonged culturing the heterogeneity is substantially lost when the melanospheres are substituted by cell aggregates. This should be considered when cell aggregates instead of melanospheres are used in the study of

  7. Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium.

    Science.gov (United States)

    Alves, A E; Padilha-Nakaghi, L C; Pires-Butler, E A; Apparicio, M; Silva, Nam; Motheo, T F; Vicente, Wrr; Luvoni, G C

    2017-04-01

    In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 μm (T0) vs. 201 ± 22.3 μm (T6); p  .05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats. © 2016 Blackwell Verlag GmbH.

  8. Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

    Science.gov (United States)

    Liu, Weiwei; Chen, Guokai

    2014-01-01

    This protocol describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. The preservation in E8 medium eliminates serum or other animal products, and is suitable for the increasing demand for high quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, this protocol allows efficient cryopreservation in more time-saving manner. PMID:25366897

  9. Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

    Science.gov (United States)

    Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

    2016-03-01

    Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = culture. Human embryo studies are needed. © The Author(s) 2015.

  10. Medium optimization for protopectinase production by batch culture ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-11

    Nov 11, 2011 ... African Journal of Biotechnology Vol. ... protopectinase production were reported to be sucrose, yeast extracts and ammonium acetate by the ... influenced by medium compositions such as carbon sources, nitrogen sources, growth factors and inorganic salts. When compared with conventional method,.

  11. Optimum culture medium composition for lipopeptide production by ...

    Indian Academy of Sciences (India)

    Central composite rotatable design (CCRD) of experiments was used to obtain data for Lipopeptide and Biomass concentrations from fermentation medium containing the following five components: glucose,monosodium glutamate, yeast extract,MgSO4·7H2O, and K2HPO4. Data was used to develop a second order ...

  12. Optimum culture medium composition for lipopeptide production by ...

    Indian Academy of Sciences (India)

    is thus shown to be an effective tool for medium optimization of biosurfactant production. Keywords. Ant colony optimization; Bacillus ... ies, media optimization is performed either by the analysis of experimental results or RSM results. ..... According to this equation, pathways with the cost function larger than C receive no trail ...

  13. Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants

    Directory of Open Access Journals (Sweden)

    Kai-Hung Wang

    2016-01-01

    Full Text Available We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40 of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

  14. Dependence of synchronized bursting activity on medium stirring and the perfusion rate in a cultured network of neurons

    Science.gov (United States)

    Heo, Ryoun; Kim, Hyun; Lee, Kyoung J.

    2016-05-01

    A cultured network of neurons coupled with a multi-electrode-array (MEA) recording system has been a useful platform for investigating various issues in neuroscience and engineering. The neural activity supported by the system can be sensitive to environmental fluctuations, for example, in the medium's nutrient composition, ph, and temperature, and to mechanical disturbances, yet this issue has not been the subject. Especially, a normal practice in maintaining neuronal cell cultures involves an intermittent sequence of medium exchanges, typically at a time interval of a few days, and one such sudden medium exchange is unavoidably accompanied by many unintended disturbances. Here, based on a quantitative time-series analysis of synchronized bursting events, we explicitly demonstrate that such a medium exchange can, indeed, bring a huge change in the existing neural activity. Subsequently, we develop a medium perfusion-stirring system and an ideal protocol that can be used in conjunction with a MEA recording system, providing long-term stability. Specifically, we systematically evaluate the effects of medium stirring and perfusion rates. Unexpectedly, even some vigorous mechanical agitations do not have any impacts on neural activity. On the other hand, too much replenishment ( e.g., 1.8 ml/day for a 1.8-ml dish) of neurobasal medium results in an excitotoxicity.

  15. An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines.

    Science.gov (United States)

    Rourou, Samia; Ben Ayed, Yousr; Trabelsi, Khaled; Majoul, Samy; Kallel, Héla

    2014-05-19

    IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

    OpenAIRE

    Uphoff, Cord C.; Denkmann, Sabine-A.; Drexler, Hans G.

    2012-01-01

    A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of...

  17. Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix

    OpenAIRE

    Kaneko, Ai; Sankai, Yoshiyuki

    2014-01-01

    The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leadin...

  18. A novel liquid medium for the efficient growth of the salmonid pathogen Piscirickettsia salmonis and optimization of culture conditions.

    Science.gov (United States)

    Henríquez, Mirtha; González, Ernesto; Marshall, Sergio H; Henríquez, Vitalia; Gómez, Fernando A; Martínez, Irene; Altamirano, Claudia

    2013-01-01

    Piscirickettsia salmonis is the bacterium that causes Piscirickettsiosis, a systemic disease of salmonid fish responsible for significant economic losses within the aquaculture industry worldwide. The growth of the bacterium for vaccine formulation has been traditionally accomplished by infecting eukaryotic cell lines, a process that involves high production costs and is time-consuming. Recent research has demonstrated that it is possible to culture pure P. salmonis in a blood containing (cell-free) medium. In the present work we demonstrate the growth of P. salmonis in a liquid medium free from blood and serum components, thus establishing a novel and simplified bacteriological medium. Additionally, the new media reported provides improved growth conditions for P. salmonis, where biomass concentrations of approximately 800 mg cell dry weight L(-1) were obtained, about eight times higher than those reported for the blood containing medium. A 2- level full factorial design was employed to evaluate the significance of the main medium components on cell growth and an optimal temperature range of 23-27°C was determined for the microorganism to grow in the novel liquid media. Therefore, these results represent a breakthrough regarding P. salmonis research in order to optimize pure P. salmonis growth in liquid blood and serum free medium.

  19. Liquid-liquid extraction and liquid chromatography-mass spectrometry detection of curcuminoids from bacterial culture medium.

    Science.gov (United States)

    Tan, Suryani; Rupasinghe, Thusitha W T; Tull, Dedreia L; Augustin, Mary Ann; Gras, Sally L

    2015-04-15

    Liquid chromatography-mass spectrometry (LC-MS) has been used to detect polyphenolic curcuminoids found in turmeric but studies of metabolism by bacterial and mammalian cells in vitro are compromised by poor recovery from the culture medium. We report a liquid-liquid extraction procedure with ethyl acetate and use LC-MS to quantify extracted curcuminoids. Ethyl acetate allows recoveries of ∼ 80-86% of curcuminoids from the bacterial growth medium, bacterial cell lysate and combined bacterial cell and growth medium matrices; a clear improvement over acetonitrile where recoveries were ∼ 25-66%. This optimised method will enable studies of curcuminoid metabolism and may be applicable to other hydrophobic polyphenolic compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

    Science.gov (United States)

    De, Arpan; Liao, Sumei; Bitoun, Jacob P; Roth, Randy; Beatty, Wandy L; Wu, Hui; Wen, Zezhang T

    2017-09-01

    Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics. Copyright © 2017 American Society for Microbiology.

  1. Studies on culture and osteogenic induction of human mesenchymal stem cells under CO2-independent conditions.

    Science.gov (United States)

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang; Wang, Jinfu

    2013-04-01

    Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the

  2. Bicarbonate Plays a Critical Role in the Generation of Cytotoxicity during SIN-1 Decomposition in Culture Medium

    Directory of Open Access Journals (Sweden)

    Kyo Shirai

    2012-01-01

    Full Text Available 3-Morpholinosydnonimine (SIN-1 is used as a donor of peroxynitrite (ONOO− in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s in the medium, rather than ONOO−, exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1.

  3. Bicarbonate Plays a Critical Role in the Generation of Cytotoxicity during SIN-1 Decomposition in Culture Medium

    Science.gov (United States)

    Shirai, Kyo; Okada, Tatsumi; Konishi, Kanako; Murata, Hiroshi; Akashi, Soichiro; Sugawara, Fumio; Watanabe, Nobuo; Arai, Takao

    2012-01-01

    3-Morpholinosydnonimine (SIN-1) is used as a donor of peroxynitrite (ONOO−) in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s) in the medium, rather than ONOO−, exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1. PMID:22848780

  4. Age of G-1 PLUS v5 embryo culture medium is inversely associated with birthweight of the newborn.

    Science.gov (United States)

    Kleijkers, Sander H M; van Montfoort, Aafke P A; Smits, Luc J M; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

    2015-06-01

    Does age of G-1 PLUS v5 embryo culture medium affect IVF outcome? Birthweight of singletons born after IVF showed an inverse association with age of the embryo culture medium, while no association was found between age of culture medium and fertilization rate, embryonic development or ongoing pregnancy. It has been reported that IVF culture media can deteriorate during storage, which suggests that the capacity of culture media to support optimal embryo development decreases over time. Some animal studies showed an effect of storage time on embryo development, in contrast to other studies, while the effect of aging culture medium on IVF outcome in humans is unknown. We used data on outcome of 1832 IVF/ICSI cycles with fresh embryo transfer, performed in the period 2008-2012 to evaluate the association of fertilization rate, embryonic development, ongoing pregnancy and birthweight of singletons with age of the culture medium (Vitrolife AB G-1 PLUS v5). Age of the culture medium was calculated by subtracting the production date from the date of ovum retrieval. Data analysis included linear regression and logistic regression on continuous and categorical outcomes, respectively. Age of the culture medium was not associated with fertilization rate (P = 0.543), early cleavage rate (P = 0.155), percentage of embryos containing four or more cells on Day 2 (P = 0.401), percentage of embryos containing eight or more cells on Day 3 (P = 0.175), percentage of embryos with multinucleated blastomeres (P = 0.527), or ongoing pregnancy (P = 0.729). However, birthweight of the newborn was inversely associated with age of the medium (β = -3.6 g, SE: 1.5 g, P = 0.021), after controlling for possible confounders (day of embryo transfer, number of transferred embryos, child's gender, gestational age at birth, parity, pregnancy complications, maternal smoking, height and weight, and paternal height and weight) and the association was not biased by year of treatment, time since first

  5. Use of secondary sewage water as a culture medium for Chaetoceros gracilis and Thalassiosira Sp (Chrysophyceae in laboratory conditions

    Directory of Open Access Journals (Sweden)

    Rauquírio André Albuquerque Marinho da Costa

    1999-01-01

    Full Text Available Experiments were carried out in order to test the efficiency of additions of secondary sewage as a culture medium for Chaetoceros gracilis and Thalassiosira sp (Chrysophyceae under laboratory conditions. These algae were cultivated in sea water with concentrations of 10%, 20%, 30% and 40% of wastewater. The results were compared with those obtained by the nutritive medium f2 of Guillard (1975. The best results in terms of cellular densities were observed at 40% additions. There were significant differences (significance levels of 5% between the nutritive medium f2 and the 40% additions for both the species. Maximum cellular densities observed for all additions tested were, 4,125.00 x 10³ cells/ml for Chaetoceros gracilis on the ninth day and 834.00 x 10³ cells/ml for Thalassiosira sp on the fifth day. Biomass was higher in the nutritive medium f2 than in the other treatments, reaching average values of 2,363μg/ml for Chaetoceros gracilis. At all experimental units, the best results were registered at 40% addition for Chaetoceros gracilis, where average values of 0.768μg/ml were observed on the fifth day, and at 30% additions for Thalassiosira sp where 0.883μg/ml were observed on the thirteenth day. It was concluded that secondary sewage could be used as a culture medium for the species tested here, after large scale tests.

  6. USE OF SODIUM HIPOCHLORITE IN STERILIZATION OF CULTURE MEDIUM FOR MULTIPLICATION OF Eucalyptus pellita L.

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    Silvio Lopes Teixeira

    2009-10-01

    Full Text Available Lately it has been observed a great interest in the research area of plant tissue culture in discovering new alternatives leading to cost reduction of the plants produced in commercial laboratories, in order to turn this alternative of plant propagation more economical. A potentially promising alternative for this reduction of costs, but which has not been receiving the due attention, is the possibility of substituting the autoclaving technique to a more economical one. With this purpose, two tests were carried out, using a new protocol of medium preparation, which consisted of the chemical sterilization of all the utensils used in the preparation and packaging of the culture medium as well, associated to the addition of the sterilizing agent to the medium, in different concentrations. The objective of the first test was to observe the influence of different concentrations of NaClO added to the culture medium, on its sterilization. The second test aimed at verifying the reaction of the Eucalyptus pellita tissues to different concentrations of NaClO in the culture medium. The addition of NaClO to the culture medium, equal or higher than 0.0005% in the fist test and of 0.005% in the second one, allowed complete sterilization of the medium, without observing any damage to the Eucalyptus pellita tissues, even when they were grown on culture medium containing up to 0.009%, the maximum concentration tried. The results showed the viability of eliminating the autoclave for the sterilization of culture media.

  7. Vertical ascending electrophoresis of cells with a minimal stabilizing medium

    Science.gov (United States)

    Omenyi, S. N.; Snyder, R. S.

    1983-01-01

    Vertical fractionation of a mixture of fixed horse and human red blood cells layered over a stabilizing support medium was done to give a valid comparison with proposed space experiments. In particular, the effects of sample thickness and concentration on zone migration rate were investigated. Electrophoretic mobilities of horse and human cells calculated from zone migration rates were compatible with those obtained by microelectrophoresis. Complete cell separation was observed when low power and effective cooling were employed.

  8. A serum-free and defined medium for the culture of mammalian postimplantation embryos.

    Science.gov (United States)

    Drakou, Katerina; Georgiades, Pantelis

    2015-12-25

    Whole embryo culture (WEC) of postimplantation rodent embryos is widely used for the study of mammalian embryogenesis and developmental toxicity testing. Its major advantage is that it allows direct access to embryos for experimental manipulations and the monitoring of their consequences that would otherwise not be possible or technically difficult to perform in utero. However, a major drawback of mammalian WEC is that the culture media currently in use display batch variations and are undefined, as they contain serum or serum replacements of unknown composition. Moreover, these media possess cell-signalling activities important for embryogenesis. Therefore, reproducibility of mammalian postimplantation WEC results may be affected by batch variation and their interpretation is complicated because the experimenter is unsure whether the embryo response to experimental perturbations is solely due to their action, or modified as a result of influences from undefined substances/signaling activities present in culture media. To alleviate these problems we investigated whether N2B27, a serum-free and defined medium, can support the in vitro development of postimplantation mammalian embryos. We show that N2B27 allows pre-gastrulation mouse embryos isolated at embryonic day 5.5 to develop to advanced gastrulation, reaching the mid- and late primitive streak stages. This is the first demonstration that postimplantation mammalian embryos can develop in vitro in a defined medium in the absence of serum and provides a novel WEC system for studying developmental mechanisms and testing for developmental toxicity during the early postimplantation period. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    Science.gov (United States)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  10. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL Adapted to a Serum-Free Medium

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    Chieh-Lun Liu

    2017-05-01

    Full Text Available Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO, cysteine sulfinate decarboxylase (CSAD, or cysteamine dioxygenase (ADO. In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

  11. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium.

    Science.gov (United States)

    Liu, Chieh-Lun; Watson, Aaron M; Place, Allen R; Jagus, Rosemary

    2017-05-25

    Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

  12. Production and secretion of a recombinant Vibrio parahaemolyticus chitinase by Escherichia coli and its purification from the culture medium.

    Science.gov (United States)

    Kadokura, Kazunari; Sakamoto, Yusuke; Saito, Kaori; Ikegami, Takanori; Hirano, Takako; Hakamata, Wataru; Oku, Tadatake; Nishio, Toshiyuki

    2007-11-01

    An open reading frame encoding the chitinase gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome. An expression plasmid containing the gene was introduced into Escherichia coli cells, and recombinant chitinase (Pa-rChi) was produced and secreted into the culture medium with the aid of the signal peptide. Pa-rChi was purified and its substrate specificity was determined.

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

    International Nuclear Information System (INIS)

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

    2004-10-01

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

  14. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.

    Science.gov (United States)

    Welch, Aaron Z; Koshland, Douglas E

    2013-12-01

    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space-saving method, amenable to high-throughput screens, for accurately measuring yeast cell viability. Copyright © 2013 John Wiley & Sons, Ltd.

  15. CULTURE OF EMBRYONIC CELLS OF DROSOPHILA MELANOGASTER IN VITRO.

    Science.gov (United States)

    HORIKAWA, M; FOX, A S

    1964-09-25

    Embryonic cells isolated from eggs ofDrosophila melanogasterhave been cultured continuously in a new medium. Generation time for cell division is 30 hours. Chromosome number remains constant for at least 10 days. Cells from embryos of the mutant maroon-like grow at the same rate as those from wild-type embryos, but cells from rosy-2 grow slower and at a lower optimum temperature.

  16. Measuring TGF-β Ligand Dynamics in Culture Medium.

    Science.gov (United States)

    Feng, Zipei; Zi, Zhike; Liu, Xuedong

    2016-01-01

    TGF-β plays an important role in a myriad of cell activities including differentiation, proliferation, and growth arrest. These effects are influenced by the concentration of TGF-β in the surrounding milieu, which is interpreted by mammalian cells and subsequently translated into meaningful signals that guide their proliferation, survival, or death. To predict cellular responses to TGF-ß signaling based on molecular mechanisms, it is important to consider how cells respond to different ligand doses and how variations in ligand exposure impact Smad signaling dynamics and subsequent gene expression. Here we describe methods to measure TGF-β concentration in the environment and approaches to perturb cellular TGF-β exposure to gain a quantitative understanding of signaling dynamics of this pathway.

  17. Optimization of an effective growth medium for culturing probiotic bacteria for applications in strict vegetarian food products

    Directory of Open Access Journals (Sweden)

    Manju Pathak

    2012-10-01

    Full Text Available Background: This study aimed to modify de Man Rogosa Sharpe culture medium (termed MRS for selective cultivation of probiotics strain for the consumption by the strictly vegetarian human population. Vegetarian probiotic foods by definition must be free from all animal-derived ingredients. This not only includes the product ingredients but the probiotic inoculum as well. Probiotic starter cultures are traditionally grown and stored in media containing milk or meatderived ingredients. The presence of these ingredients makes the probiotic cell concentrates unsuitable for use in vegetarian products and thus creates the need for a growth medium which isfree from animal-derived ingredients. Present study investigated the growth of a strain of Lactobacillus lactis in MRS. The present invention relates in general to a bacterial culture media,and more specifically a complex microbial culture media, based on plant seed powder extract in place of animal extract for probiotic bacterial growth.Methods: Lactobacillus lactis, a probiotic, was grown in standard MRS culture medium as well as in our various test media (TM containing various vegetal source in place of beef extract, yeast extract and peptone as in case of MRS. The inoculated culture mediums were incubated at 37C for 72 hours and growth of probiotic is recorded at regular intervals. The growth was recorded as Colony Forming Units (CFUs.Results: The best growth of probiotic is observed in TM 2. TM 2 is the leguminous seed extract. Starter culture mediums for probiotics or other bacteria primarily contain protein from animal source. The possibility of using vegetal protein from TM 2 extract in place of peptones and meat extract for the nitrogen supplementation of culture media for the growth of lactic acid bacteria has been demonstrated.Functional Foods in Health and Disease 2012, 2(10:369-378 Conclusion: The absolute vegetarian culture medium containing TM 2 is better than standard MRS for the

  18. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

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

  19. 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.

  20. Acetaldehyde and hexanaldehyde from cultured white cells

    Directory of Open Access Journals (Sweden)

    Zaldivar Frank

    2009-04-01

    Full Text Available Abstract Background Noninvasive detection of innate immune function such as the accumulation of neutrophils remains a challenge in many areas of clinical medicine. We hypothesized that granulocytes could generate volatile organic compounds. Methods To begin to test this, we developed a bioreactor and analytical GC-MS system to accurately identify and quantify gases in trace concentrations (parts per billion emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, frequently used to assess neutrophil function, was grown in serum-free medium. Results HL60 cells released acetaldehyde and hexanaldehyde in a time-dependent manner. The mean ± SD concentration of acetaldehyde in the headspace above the cultured cells following 4-, 24- and 48-h incubation was 157 ± 13 ppbv, 490 ± 99 ppbv, 698 ± 87 ppbv. For hexanaldehyde these values were 1 ± 0.3 ppbv, 8 ± 2 ppbv, and 11 ± 2 ppbv. In addition, our experimental system permitted us to identify confounding trace gas contaminants such as styrene. Conclusion This study demonstrates that human immune cells known to mimic the function of innate immune cells, like neutrophils, produce volatile gases that can be measured in vitro in trace amounts.

  1. Buffering the pH of the culture medium does not extend yeast replicative lifespan [v1; ref status: indexed, http://f1000r.es/20w

    OpenAIRE

    Brian M Wasko; Daniel T Carr; Herman Tung; Ha Doan; Nathan Schurman; Jillian R Neault; Joey Feng; Janet Lee; Ben Zipkin; Jacob Mouser; Edward Oudanonh; Tina Nguyen; Torin Stetina; Anna Shemorry; Mekedes Lemma

    2013-01-01

    During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival.  As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium.  In this study, we assessed whether a similar process occurs during replicative aging of yeast cells.  We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the...

  2. In vitro differentiation of mouse embryonic stem cells into inner ear hair cell-like cells using stromal cell conditioned medium.

    Science.gov (United States)

    Ouji, Y; Ishizaka, S; Nakamura-Uchiyama, F; Yoshikawa, M

    2012-05-24

    Hearing loss is mainly caused by loss of sensory hair cells (HCs) in the organ of Corti or cochlea. Although embryonic stem (ES) cells are a promising source for cell therapy, little is known about the efficient generation of HC-like cells from ES cells. In the present study, we developed a single-medium culture method for growing embryoid bodies (EBs), in which conditioned medium (CM) from cultures of ST2 stromal cells (ST2-CM) was used for 14-day cultures of 4-day EBs. At the end of the 14-day cultures, up to 20% of the cells in EB outgrowths expressed HC-related markers, including Math1 (also known as Atoh1), myosin6, myosin7a, calretinin, α9AchR and Brn3c (also known as Pou4f3), and also showed formation of stereocilia-like structures. Further, we found that these cells were incorporated into the developing inner ear after transplantation into chick embryos. The present inner ear HC induction method using ST2-CM (HIST2 method) is quite simple and highly efficient to obtain ES-derived HC-like cells with a relatively short cultivation time.

  3. Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

    2012-01-01

    Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

  4. STUDIES REGARDING CULTURE MEDIUM INFLUENCE ON IN VITRO REGENERATION FROM WHEAT IMATURE EMBRYOS

    Directory of Open Access Journals (Sweden)

    M. DANCI

    2008-05-01

    Full Text Available Surnamed „embryos’ saving method”, embryos culture is an in vitro technique used for over half of the century for saving the distant hybridization products, that would have degenerate in other conditions. Immature embryos culture is used for initiation of in vitro cultures imposed by the impossibility of using other explants for some of the plant species. Wheat is one of the crops that immature embryos culture technique is suitable for. This methods principle is based on aseptic embryos excision and their inoculation to an adequate culture medium. In vitro culture results depend in a greater manner of the basic culture medium and the hormonal balance used. Immature embryos isolated from two Romanian wheat cultivars – Dropia and Lovrin 41 – were inoculated for callus production on two types of basic media added with 2,4 D. The selected calluses were transferred on MS basic medium and several parameters were registered. Both cultivars emphasized a good callusing capacity, in a different percentage depending on the culture media used, such as 71,09 – 94,45%.. big differences between the cultivars regarding embriogenic callus frequency, shooting callus frequency and regenerated plants percentage were registered.

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

    Science.gov (United States)

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

    2015-08-01

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

  6. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  7. Fermentation profile of Saccharomyces cerevisiae and Candida tropicalis as starter cultures on barley malt medium.

    Science.gov (United States)

    Alloue-Boraud, Wazé Aimée Mireille; N'Guessan, Kouadio Florent; Djeni, N'Dédé Théodore; Hiligsmann, Serge; Djè, Koffi Marcellin; Delvigne, Franck

    2015-08-01

    Saccharomyces cerevisiae C8-5 and Candida tropicalis F0-5 isolated from traditional sorghum beer were tested for kinetic parameters on barley malt extract, YPD (863 medium) and for alcohol production. The results showed that C. tropicalis has the highest maximum growth rate and the lowest doubling time. Values were 0.22 and 0.32 h(-1) for maximum growth rate, 3 h 09 min and 2 h 09 min for doubling time respectively on barley malt extract and YPD. On contrary, glucose consumption was the fastest with S. cerevisiae (-0.36 and -0.722 g/l/h respectively on barley malt extract and YPD). When these two yeasts were used as starters in pure culture and co-culture at proportion of 1:1 and 2:1 (cell/cell) for barley malt extract fermentation, we noticed that maltose content increased first from 12.12 g/l to 13.62-16.46 g/l and then decreased. The highest increase was obtained with starter C. tropicalis + S. cerevisiae 2:1. On contrary, glucose content decreased throughout all the fermentation process. For all the starters used, the major part of the ethanol was produced at 16 h of fermentation. Values obtained in the final beers were 11.4, 11.6, 10.4 and 10.9 g/l for fermentation conducted with S. cerevisiae, C. tropicalis, C. tropicalis + S. cerevisiae 1:1 and C. tropicalis + S. cerevisiae 2:1. Cell viability measurement during the fermentation by using flow cytometry revealed that the lowest mean channel fluorescence for FL3 (yeast rate of death) was obtained with C. tropicalis + S. cerevisiae 2:1 after 48 h of fermentation.

  8. [Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

    Science.gov (United States)

    Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang

    2008-07-01

    In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.

  9. Embryo quality and implantation rate in two different culture media: ISM1 versus Universal IVF Medium.

    Science.gov (United States)

    Xella, Susanna; Marsella, Tiziana; Tagliasacchi, Daniela; Giulini, Simone; La Marca, Antonio; Tirelli, Alessandra; Volpe, Annibale

    2010-04-01

    To compare the outcome of two different culture media marketed by the MediCult AS Company (Jyllinge, Denmark)-Universal IVF Medium and ISM1 Medium culture-which, in addition to glucose, pyruvate, and energy-providing components, also contain amino acids, nucleotides, vitamins, and cholesterol. Laboratory and retrospective clinical study. University teaching hospital. A total of 726 patients, undergoing IVF-intracytoplasmic sperm injection procedure, comparable in mean age range, oocyte retrieval, and infertility indication, were included in the study. Laboratory quality and standard procedures were maintained unaffected. Oocyte retrieval, different embryo culture media. Embryo quality, ongoing pregnancy, and implantation rate. The frequency of good-quality embryos (79% vs. 74%) and the percentages of ongoing pregnancy (27.5% vs. 18%) and implantation rate (15% vs. 10%) were significantly higher in the group treated with ISM1 Medium rather than Universal IVF Medium. ISM1 Medium culture seems to improve the performance of embryonic growth and development, as well as increasing the percentage of pregnancy. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Pluronic polyols in human lymphocyte cell line cultures.

    Science.gov (United States)

    Mizrahi, A

    1975-01-01

    Pluronic polyols markedly improved the growth of two human lymphocyte cell lines when added to the growth medium in concentrations of 0.05 to 0.1%. The results of the current studies suggest that, in addition to the protective effect of polyols against mechanical damage of mammalian cells in submerged cultures, the pluronic compounds may also, by lowering surface tension, facilitate transport of metabolites into cells and thus increase the growth rate. PMID:1063740

  11. Dose verification by OSLDs in the irradiation of cell cultures

    International Nuclear Information System (INIS)

    Meca C, E. A.; Bourel, V.; Notcovich, C.; Duran, H.

    2015-10-01

    The determination of value of irradiation dose presents difficulties when targets are irradiated located in regions where electronic equilibrium of charged particle is not reached, as in the case of irradiation -in vitro- of cell lines monolayer-cultured, in culture dishes or flasks covered with culture medium. The present study aimed to implement a methodology for dose verification in irradiation of cells in culture media by optically stimulated luminescence dosimetry (OSLD). For the determination of the absorbed dose in terms of cell proliferation OSL dosimeters of aluminum oxide doped with carbon (Al 2 O 3 :C) were used, which were calibrated to the irradiation conditions of culture medium and at doses that ranged from 0.1 to 15 Gy obtained with a linear accelerator of 6 MV photons. Intercomparison measurements were performed with an ionization chamber of 6 cm 3 . Different geometries were evaluated by varying the thicknesses of solid water, air and cell culture medium. The results showed deviations below 2.2% when compared with the obtained doses of OSLDs and planning system used. Also deviations were observed below 3.4% by eccentric points of the irradiation plane, finding homogeneous dose distribution. Uncertainty in the readings was less than 2%. The proposed methodology contributes a contribution in the dose verification in this type of irradiations, eliminating from the calculation uncertainties, potential errors in settling irradiation or possible equipment failure with which is radiating. It also provides certainty about the survival curves to be plotted with the experimental data. (Author)

  12. Utilization and optimization of a waste stream cellulose culture medium for pigment production by Penicillium spp.

    Science.gov (United States)

    Sopandi, T; Wardah, A; Surtiningsih, T; Suwandi, A; Smith, J J

    2013-03-01

    This research sought to determine optimal corn waste stream-based fermentation medium C and N sources and incubation time to maximize pigment production by an indigenous Indonesian Penicillium spp., as well as to assess pigment pH stability. A Penicillium spp. was isolated from Indonesian soil, identified as Penicillium resticulosum, and used to test the effects of carbon and nitrogen type and concentrations, medium pH, incubation period and furfural on biomass and pigment yield (PY) in a waste corncob hydrolysate basal medium. Maximum red PY (497.03 ± 55.13 mg l(-1)) was obtained with a 21 : 1 C : N ratio, pH 5.5-6.0; yeast extract-, NH(4) NO(3)-, NaNO(3)-, MgSO(4) ·7H(2) O-, xylose- or carboxymethylcellulose (CMC)-supplemented medium and 12 days (25 °C, 60-70% relative humidity, dark) incubation. C source, C, N and furfural concentration, medium pH and incubation period all influenced biomass and PY. Pigment was pH 2-9 stable. Penicillium resticulosum demonstrated microbial pH-stable-pigment production potential using a xylose or CMC and N source, supplemented waste stream cellulose culture medium. Corn derived, waste stream cellulose can be used as a culture medium for fungal pigment production. Such application provides a process for agricultural waste stream resource reuse for production of compounds in increasing demand. © 2012 The Society for Applied Microbiology.

  13. Development of culture medium using extruded bean as a nitrogen source for yeast growth.

    Science.gov (United States)

    Batista, Karla A; Bataus, Luiz Artur M; Campos, Ivan T N; Fernandes, Kátia F

    2013-03-01

    In this study extruded bean was used as a nitrogen source substitute in culture medium formulation. A 3-factor simplex-lattice mixture design was used to establish better growth conditions. Completely substituted medium resulted in 43% of increase in the growth of Saccharomyces cerevisiae. Mixtures containing 1% extruded bean and 1% yeast extract, or 1% extruded bean and 1% peptone presented growths of 76-79% higher than the commercial YPD medium for S. cerevisiae. Pichia pastoris (GS115) growth was enhanced by 20% using a completely substituted medium. The protein expression patterns in P. pastoris (GS115) remained unchanged when growth was conducted in a medium containing extruded bean as unique nitrogen source. The total amount of recombinant protein expressed in extruded bean medium was 88.5% higher than in control expression medium. These results evidenced that extruded bean can be successfully used as a substitute of peptone and yeast extract in culture media for S. cerevisiae's and P. pastoris' (GS115) growth. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. [Culture medium based on biogas slurry and breeding of oil Chlorella].

    Science.gov (United States)

    Zhao, Feng-Min; Mei, Shuai; Cao, You-Fu; Ding, Jin-Feng; Xu, Jia-Jie; Li, Shu-Jun

    2014-06-01

    The oil chlorella cultivation and biogas slurry treatment were combined. The biogas slurry provided water and nutrient for growing chlorella, at the same time, harmless treatment of biogas slurry was realized. This paper cultivated 4 species of oil chlorella in the mixed medium of biogas slurry and green algae medium (the volume ratios were 1 : 9, 1 : 3, 1 : 1 and 3 : 1, respectively), and compared their oil productivity to select the best oil chlorella species and the optimal culture medium. The results showed that, the combination of medium and chlorella species to reach the highest oil productivity was a volume ratio of 1 : 3 and the chlorella species BJ05, and the oil productivity of chlorella BJ05 was 9.20 mg x (L x d)(-1), higher than that in green algae medium [8.66 mg x (L x d)(-1)]. In mixed medium with a volume ratio of 1:3, the effect of adding different nutrients into the green algae medium on the oil productivity was examined, and the results showed that, sodium carbonate and citric acid had no negative effect on the oil productivity of chlorella BJ05. in the absence of sodium carbonate and citric acid, the oil productivity of chlorella BJ05 was 9.36 mg x (L x d)(-1), and the removal of COD (chemical oxygen demand), total nitrogen, total phosphorus and ammonia nitrogen rates were 59%, 75%, 61% and 100%, respectively. Deficiency in other nutrients had negative effect on the oil productivity. Therefore, the culture medium was further optimized to the mixed medium of biogas slurry and green algae medium with a volume ratio of 1 : 3 and without addition of sodium carbonate and citric acid.

  15. Youth Culture and Cell Phone

    Directory of Open Access Journals (Sweden)

    mohammad saeed zokaei

    2009-11-01

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

  16. Buffering the pH of the culture medium does not extend yeast replicative lifespan [v1; ref status: indexed, http://f1000r.es/20w

    Directory of Open Access Journals (Sweden)

    Brian M Wasko

    2013-10-01

    Full Text Available During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival.  As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium.  In this study, we assessed whether a similar process occurs during replicative aging of yeast cells.  We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan.  Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.

  17. The effect of supplementation of amino acids and taurine to modified KSOM culture medium on rat embryo development.

    Science.gov (United States)

    Nakamura, Kazuomi; Morimoto, Kayoko; Shima, Kaoru; Yoshimura, Yuki; Kazuki, Yasuhiro; Suzuki, Osamu; Matsuda, Junichiro; Ohbayashi, Tetsuya

    2016-11-01

    The rat is widely used as a laboratory animal for research. In particular, genetically engineered rats are essential for production of animal models of several diseases. Although embryo manipulation techniques are needed to produce them, such technology for rat preimplantation embryos is not as advanced as it is for mouse embryos. One reason is that in vitro culture systems for preimplantation embryos are limited in rats. Therefore, we intended to develop a new culture system for rat preimplantation embryos focusing on supplementation of amino acids as nutrition to the culture media. First, we found that taurine, glycine, glutamate, and alanine were abundant in the oviductal fluid of Wistar rats. The profile of taurine and these three amino acids was unchanged during the estrous cycle and from Days 0 to 3 of pregnancy (Day 0; vaginal plug was confirmed). Second, we assessed the effect of phosphate and phenol red on the development of rat zygotes and confirmed that they caused two-cell block. Third, we examined the effect of changing the medium on zygote development because addition of amino acids into culture medium causes ammonium accumulation, which is detrimental to embryo development. Blastocyst formation was suppressed in cultures with no medium change (P = 0.004; decreased to approximately one-fourth of that with medium change). Fourth, we examined the effect of supplementation of these three amino acids and taurine to modified potassium simplex optimized medium (KSOM). The zygote development rates were increased by the three amino acids and taurine in a concentration-dependent manner at 48, 72, and 96 hours (P = 0.001, 0.005, and 0.009, respectively) in culture. Finally, we confirmed that blastocysts cultured in modified KSOM had the capacity to develop to full term after implantation. These results showed that not only the supply of nutrients but also removal of wastes and toxicants is important for culture of rat preimplantation embryos. Copyright

  18. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  19. Bystander Effects Induced by Medium From Irradiated Cells: Similar Transcriptome Responses in Irradiated and Bystander K562 Cells

    International Nuclear Information System (INIS)

    Herok, Robert; Konopacka, Maria; Polanska, Joanna; Swierniak, Andrzej; Rogolinski, Jacek; Jaksik, Roman; Hancock, Ronald; Rzeszowska-Wolny, Joanna

    2010-01-01

    Purpose: Cells exposed to ionizing radiation release factors that induce deoxyribonucleic acid damage, chromosomal instability, apoptosis, and changes in the proliferation rate of neighboring unexposed cells, phenomena known as bystander effects. This work analyzes and compares changes in global transcript levels induced by direct irradiation and by bystander effects in K562 (human erythroleukemia) cells. Methods and Materials: Cells were X-irradiated with 4 Gy or transferred into culture medium collected from cells 1 h after irradiation (irradiation-conditioned medium). Global transcript profiles were assessed after 36 h of growth by use of Affymetrix microarrays (Affymetrix, Santa Clara, CA) and the kinetics of change of selected transcripts by quantitative reverse transcriptase-polymerase chain reaction. Results: The level of the majority (72%) of transcripts changed similarly (increase, decrease, or no change) in cells grown in irradiation-conditioned medium or irradiated, whereas only 0.6% showed an opposite response. Transcript level changes in bystander and irradiated cells were significantly different from those in untreated cells grown for the same amount of time and were confirmed by quantitative reverse transcriptase-polymerase chain reaction for selected genes. Signaling pathways in which the highest number of transcripts changed in both conditions were found in the following groups: neuroactive ligand-receptor, cytokine-cytokine receptor interaction, Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) and Mitogen-Activated Protein Kinase (MAPK) In control cells more transcripts were downregulated than in irradiated and bystander cells with transcription factors YBX1 and STAT5B, heat shock protein HSPA1A, and ribonucleic acid helicase DDX3X as examples. Conclusions: The transcriptomes of cells grown in medium from X-irradiated cells or directly irradiated show very similar changes. Signals released by irradiated cells may cause

  20. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice.

    Directory of Open Access Journals (Sweden)

    Lei Chen

    Full Text Available Growing evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs enhance wound repair via paracrine. Because the extent of environmental oxygenation affects the innate characteristics of BM-MSCs, including their stemness and migration capacity, the current study set out to elucidate and compare the impact of normoxic and hypoxic cell-culture conditions on the expression and secretion of BM-MSC-derived paracrine molecules (e.g., cytokines, growth factors and chemokines that hypothetically contribute to cutaneous wound healing in vivo. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA analyses of normoxic and hypoxic BM-MSCs and their conditioned medium fractions showed that the stem cells expressed and secreted significantly higher amounts of basic fibroblast growth factor (bFGF,vascular endothelial growth factor A (VEGF-A interleukin 6 (IL-6 and interleukin 8 (IL-8 under hypoxic conditions. Moreover, hypoxic BM-MSC-derived conditioned medium (hypoCM vs. normoxic BM-MSC-derived conditioned medium (norCM or vehicle control medium significantly enhanced the proliferation of keratinocytes, fibroblasts and endothelial cells, the migration of keratinocytes, fibroblasts, endothelial cells and monocytes, and the formation of tubular structures by endothelial cells cultured on Matrigel matrix. Consistent with these in vitro results, skin wound contraction was significantly accelerated in Balb/c nude mice treated with topical hypoCM relative to norCM or the vehicle control. Notably increased in vivo cell proliferation, neovascularization as well as recruitment of inflammatory macrophages and evidently decreased collagen I, and collagen III were also found in the hypoCM-treated group. These findings suggest that BM-MSCs promote murine skin wound healing via hypoxia-enhanced paracrine.

  1. Determination of thymidine in serum used for cell culture media

    International Nuclear Information System (INIS)

    Schaer, J.C.; Maurer, U.; Schindler, R.

    1978-01-01

    Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

  2. Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

    Directory of Open Access Journals (Sweden)

    Torres P.M.M.

    1998-01-01

    Full Text Available Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC. These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

  3. GlutaMAX prolongs the shelf life of the culture medium for porcine parthenotes.

    Science.gov (United States)

    Zhao, Ming-Hui; Kim, Nam-Hyung; Cui, Xiang-Shun

    2016-02-01

    In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. MULTISECTORAL MODELS FOR ADVANCED KNOWLEDGE IN CULTURE AND SPORTS ORGANIZATIONAL ENTITIES, IN SMALL AND MEDIUM COMPANIES

    OpenAIRE

    Mihai-Daian, CEAUSU

    2015-01-01

    The article used to identify functional and operational links, and determining the quasi-unlimited ecoknowledge in organizational culture and sports entities, in small and medium firms. It is shown that, in fact, increased in absolute economic indicators of ontemporary productive firms, in organizational culture and sports entities explained increasingly by innovation, springing from some new features, on specific contemporary environment organized / structured in corporate regime, by functi...

  5. Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome.

    Science.gov (United States)

    Wirleitner, B; Vanderzwalmen, P; Stecher, A; Zech, M H; Zintz, M; Zech, N H

    2010-12-01

    The elucidation of the metabolic requirements of human embryos in vivo or in vitro remains, despite being intensively investigated, a work in progress. The adoption of extended embryo culture to the blastocyst stage during the last decade has entailed new challenges. With the increased attention to culture media formulations, more evidence on the sensitivity of embryos to their early environmental conditions is accumulating which might affect phenotype and developmental potential. A retrospective study was conducted that comprised 286 IVF cycles to evaluate the effect of two different culture media on blastocyst development and pregnancy outcome. Embryos were either cultured in a one step or a sequential medium. Higher fertilization rates and augmented blastocyst rates as well as higher implantation rates were observed when embryos were cultured in one step medium (Pcultured in either medium resulted in a significantly higher rate of twin pregnancies. Although multiple pregnancies should be avoided in assisted reproduction treatment to reduce risks for offspring and mother, this higher frequency of twin pregnancies resulting from the transfer of embryos derived from different culture media suggests that each embryo makes individual demands on its early environment. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  6. Comparative evaluation of chromogenic agar medium and conventional culture system for isolation and presumptive identification of uropathogens.

    Science.gov (United States)

    Akter, Laila; Haque, Rezwana; Salam, Md Abdus

    2014-09-01

    Urine is the most frequent specimen received for culture/sensitivity by clinical laboratories. The microbiological performance of HiCrome UTI agar medium was compared with Blood agar and MacConkey agar for isolation and presumptive identification of bacteria from urine culture. A total of 443 consecutively collected midstream and/or catheter-catch urine samples from patients attending the Islami Bank Medical College Hospital, Rajshahi, Bangladesh during January to December, 2012 were cultured. Urine samples showing pus cells ≥ 5/HPF were inoculated on to Blood agar (BA), MacConkey agar (MAC) and HiCrome UTI agar (CA) media simultaneously and incubated overnight aerobically at 37(0)C. Rate of isolation and presumptive identification of bacterial species were compared for different media. Culture yielded a total of 199 bacterial isolates from 189 (42.67%) positive plates including 179 (40.40%) unimicrobial and 10 (2.26%) polymicrobial (mixed growth of pair of bacteria) growths. Both HiCrome UTI agar and Blood agar media supported 100% growths while 151 (75.88%) growths were observed on MacConkey agar. The rate of presumptive identification was found significantly higher on HiCrome UTI agar (97.49%) than MAC agar (67.34%) (P<0.001) as primary urine culture medium. Of 199 isolates, E. coli was found to be the leading uropathogen isolated from 118 (59.30%) samples with its presumptive identification rate of 95.76%, 93.22% and 5.93% on CA, MAC and BA respectively. All 10 (100%) polymicrobial growths were demonstrated distinctly on CA against only 01(10%) on each BA and MAC. HiCrome UTI agar was found to be more useful as primary urine culture medium in both higher rate of isolation and presumptive identification of uropathogens in comparison to conventional media. Its inherent characteristics in demonstrating polymicrobial growth and ease of rapid identification by distinct colony colour are unique.

  7. 21 CFR 866.2410 - Culture medium for pathogenic Neisseria spp.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Culture medium for pathogenic Neisseria spp. 866.2410 Section 866.2410 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... belonging to the genus Neisseria, such as epidemic cerebrospinal meningitis, other meningococcal disease...

  8. English Literature at English-Medium Schools of Bangladesh: The Question of Culture

    Science.gov (United States)

    Al-Quaderi, Golam Gaus; Al Mahmud, Abdullah

    2010-01-01

    English-medium schools have been in existence in Bangladesh since the time of British colonial rule and English literature has been part of the curriculum almost from their inception. Considered to be the carrier of the values, culture and worldview of the colonisers in the colonial era, it is today connected with the neo-colonial world…

  9. Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

    Directory of Open Access Journals (Sweden)

    Márcia Aiko Shirakawa

    2011-06-01

    Full Text Available The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS, as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96% of the calcium, this strain thus has good potential for use on building materials.

  10. Emerging Culture of English-Medium Instruction in Korea: Experiences of Korean and International Students

    Science.gov (United States)

    Kim, Jeongyeon; Tatar, Bradley; Choi, Jinsook

    2014-01-01

    This study aims to contrastively examine Korean and international students' experiences of taking subject courses at a Korean university. Focusing on the viewpoints of the students, rather than central authorities, we attempt to reveal how language use and cultural factors are interpenetrated in the praxis of English-medium instruction (EMI). The…

  11. Liquid culture medium for the rapid cultivation of Helicobacter pylori from biopsy specimens.

    Science.gov (United States)

    Sainsus, N; Cattori, V; Lepadatu, C; Hofmann-Lehmann, R

    2008-12-01

    The goal of this study was to develop a liquid culture medium for the rapid isolation, cultivation, identification and subsequent antibiotics susceptibility testing of Helicobacter pylori directly from biopsy specimens. Five liquid media were tested: Ham's F-12, Brucella broth, tryptic soybroth, brain heart infusion broth and Mueller-Hinton broth. After optimisation of the medium, it was applied in order to investigate biopsy samples from 150 patients with gastro-duodenal disorders and compared with traditional culture methods, microscopy and an H. pylori-specific TaqMan real-time polymerase chain reaction (PCR). The most reliable and rapid growth of H. pylori, even at a small inoculum size, was obtained in Ham's F-12 medium with 5% horse serum. The developed system allowed the primary isolation of H. pylori in clinical samples and provided 87% sensitivity and 100% specificity.

  12. Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

    Science.gov (United States)

    Whittington, Richard J; Whittington, Ann-Michele; Waldron, Anna; Begg, Douglas J; de Silva, Kumi; Purdie, Auriol C; Plain, Karren M

    2013-12-01

    Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 μl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

  13. Selection and physiology of cell cultures of Douglas-fir grown under conditions of water stress.

    Science.gov (United States)

    Leustek, T; Kirby, E G

    1990-09-01

    Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) cell cultures sampled 3, 6, or 9 days after subculture in nutrient medium were able to survive subsequent subculture in a medium containing 15% polyethylene glycol (PEG) (M(r) 6000-8000) (-1.21 MPa), whereas cell sampled 12 or 16 days after subculture in nutrient medium became senescent when transferred to a medium containing 15% PEG. Cells sampled after subculture for 3, 6, or 9 days in nutrient medium had lower fresh weight/dry weight ratios, lower osmotic potentials, smaller cell diameters, and higher turgor pressures than cells sampled after 12 or 16 days subculture in nutrient medium. Cells surviving subculture to a medium containing 15% PEG did not increase in dry weight for 5 weeks even though the medium was exchanged every 7 days. After 5 weeks, however, dry weight growth resumed and reached 75% of the level attained by control cells grown on PEG-free medium. Long-term growth on a medium containing 15% PEG (PEG-selected cells) could only be sustained if the medium was supplemented with 30 mM glutamine. The PEG-selected cells grew in small clusters, were isodiametric, and had chlorophyll contents 50% higher than unselected cells. The PEG-selected cells also showed lowered cellular osmotic potentials, presumably due to osmoregulation. Turgor pressures of PEG-selected cells were greater than or equal to those of unselected cells.

  14. Growth and Development of Colletotrichum gloeosporioides f. alatae During Culture in Liquid Medium

    Directory of Open Access Journals (Sweden)

    Laura E. Cerón Rincón

    2006-01-01

    Full Text Available Some characteristics known as virulence factors for Colletotrichum sp. genus, like: weight of the produced mycelium, sporulation, poligalacturonase activity and pH medium were evaluated during the growth of C. gloeosporioides f. alatae in three liquid medium commonly used for fungi culture (Czapeck, Martin broth and potato broth and additionally (Czapeck with yam extract as the only source of carbon. After of 17 days of growth, maximum values were obtained for the above parameters in the last medium, compared with others growth media evaluated. The implemented medium with yam extract, supply nutritional requirements of the pathogen for the development of characteristic factors related with mechanism of infections that may play a role in the pathogenesis.

  15. Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

    Science.gov (United States)

    Santana, Priscila Di Paula Bessa; Silva, Thiago Velasco Guimarães; da Costa, Nathália Nogueira; da Silva, Bruno Barauna; Carter, Timothy Frederick; Cordeiro, Marcela da Silva; da Silva, Bruno José Martins; Santos, Simone do Socorro Damasceno; Herculano, Anderson Manoel; Adona, Paulo Roberto; Ohashi, Otávio Mitio; Miranda, Moysés dos Santos

    2014-10-01

    Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P L-arginine decreased blastocyst rates (P L-arginine improved embryo hatching rates (P supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos. © 2014 Wiley Periodicals, Inc.

  16. A kinetic model for flavonoid production in tea cell culture.

    Science.gov (United States)

    Shibasaki-Kitakawa, Naomi; Iizuka, Yasuhiro; Takahashi, Atsushi; Yonemoto, Toshikuni

    2017-02-01

    As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

  17. Does Embryo Culture Medium Influence the Health and Development of Children Born after In Vitro Fertilization?

    Science.gov (United States)

    Bouillon, Céline; Léandri, Roger; Desch, Laurent; Ernst, Alexandra; Bruno, Céline; Cerf, Charline; Chiron, Alexandra; Souchay, Céline; Burguet, Antoine; Jimenez, Clément; Sagot, Paul; Fauque, Patricia

    2016-01-01

    In animal studies, extensive data revealed the influence of culture medium on embryonic development, foetal growth and the behaviour of offspring. However, this impact has never been investigated in humans. For the first time, we investigated in depth the effects of embryo culture media on health, growth and development of infants conceived by In Vitro Fertilization until the age of 5 years old. This single-centre cohort study was based on an earlier randomized study. During six months, in vitro fertilization attempts (No. 371) were randomized according to two media (Single Step Medium—SSM group) or Global medium (Global group). This randomized study was stopped prematurely as significantly lower pregnancy and implantation rates were observed in the SSM group. Singletons (No. 73) conceived in the randomized study were included (42 for Global and 31 for SSM). The medical data for gestational, neonatal and early childhood periods were extracted from medical records and parental interviews (256 variables recorded). The developmental profiles of the children in eight domains (social, self-help, gross motor, fine motor, expressive language, language comprehension, letter knowledge and number knowledge – 270 items) were compared in relation to the culture medium. The delivery rate was significantly lower in the SSM group than in the Global group (p<0.05). The culture medium had no significant effect on birthweight, risk of malformation (minor and major), growth and the frequency of medical concerns. However, the children of the Global group were less likely than those of the SSM group to show developmental problems (p = 0.002), irrespective of the different domains. In conclusion, our findings showed that the embryo culture medium may have an impact on further development. PMID:27008092

  18. Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

    Science.gov (United States)

    Chou, Ya-Shuan; Young, Tai-Horng; Lou, Pei-Jen

    2015-11-01

    Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the

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

    Science.gov (United States)

    Imoto, Setsuko; Ohta, Yoshimoto

    1988-01-01

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

  20. Addition of Carbon to the Culture Medium Improves the Detection Efficiency of Aflatoxin Synthetic Fungi

    Directory of Open Access Journals (Sweden)

    Tadahiro Suzuki

    2016-11-01

    Full Text Available Aflatoxin (AF is a harmful secondary metabolite that is synthesized by the Aspergillus species. Although AF detection techniques have been developed, techniques for detection of AF synthetic fungi are still required. Techniques such as plate culture methods are continually being modified for this purpose. However, plate culture methods require refinement because they suffer from several issues. In this study, activated charcoal powder (carbon was added to a culture medium containing cyclodextrin (CD to enhance the contrast of fluorescence and improve the detection efficiency for AF synthetic fungi. Two culture media, potato dextrose agar and yeast extract sucrose agar, were investigated using both plate and liquid cultures. The final concentrations of CD and carbon in the media were 3 mg/mL and 0.3 mg/mL, respectively. Addition of carbon improved the visibility of fluorescence by attenuating approximately 30% of light scattering. Several fungi that could not be detected with only CD in the medium were detected with carbon addition. The carbon also facilitated fungal growth in the potato dextrose liquid medium. The results suggest that addition of carbon to media can enhance the observation of AF-derived fluorescence.

  1. Defined medium for Aquaspirillum serpens VHL effective in batch and continuous culture.

    Science.gov (United States)

    Whitby, G E; Murray, R G

    1980-01-01

    A defined medium for Aquaspirillum serpens VHL allows the replacement of the complex media now in use. It was developed by batch culture methods but supports growth in continuous culture. A basal salts medium supplemented with L-aspartic acid, L-alanine, and L-glutamic acid provided the best growth (turbidity), as long as ammonium chloride was omitted. Ammonium chloride caused either a lag or a reduction or a complete inhibition of the growth of A. serpens VHL on the above amino acids and other organic supplements depending on the combination used. Ammonium sulfate and ammonium hydroxide with L-glutamic acid allowed growth, but the lag period was increased in shake flask cultures. Vitamins, cysteine hydrochloride, and carbon dioxide had no effect on the growth rate. Viability (less than 50%) was inadequate to maintain continuous culture with L-glutamic acid as the sole source of carbon and nitrogen. Combinations of amino and carboxylic acids were then tested and, of these, L-glutamic acid (1 g/liter) and L-histidine (75 mg/liter) without ammonium chloride in the basal salts medium supported growth in batch and continuous culture. L-Glutamic acid was the limiting substrate for growth.

  2. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong, E-mail: nzhang@fudan.edu.cn

    2013-11-15

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

  3. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    International Nuclear Information System (INIS)

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

    2013-01-01

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC

  4. A protease/peptidase from culture medium of Flammulina velutipes that acts on arabinogalactan-protein.

    Science.gov (United States)

    Yoshimi, Yoshihisa; Sugawara, Yumi; Hori, Chiaki; Igarashi, Kiyohiko; Kaneko, Satoshi; Tsumuraya, Yoichi; Kotake, Toshihisa

    2017-03-01

    Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.

  5. Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow.

    Science.gov (United States)

    Meinel, Lorenz; Karageorgiou, Vassilis; Fajardo, Robert; Snyder, Brian; Shinde-Patil, Vivek; Zichner, Ludwig; Kaplan, David; Langer, Robert; Vunjak-Novakovic, Gordana

    2004-01-01

    We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.

  6. Production of Medium Chain Length Polyhydroxyalkanoates From Oleic Acid Using Pseudomonas putida PGA1 by Fed Batch Culture

    Directory of Open Access Journals (Sweden)

    Sidik Marsudi

    2010-10-01

    Full Text Available Bacterial polyhydroxyalkanoates (PHAs are a class of p0lymers currently receiving much attention because of their potential as renewable and biodegradable plastics. A wide variety of bacteria has been reported to produce PHAs including Pseudomonas strains. These strains are known as versatile medium chain length PHAs (PHAs-mcl producers using fatty acids as carbon source. Oleic acid was used to produce PHAs-mcl using Pseudomonas putida PGA 1 by continuous feeding of both nitrogen and carbon source, in a fed batch culture. During cell growth, PHAs also accumulated, indicating that PHA production in this organism is growth associated. Residual cell increased until the nitrogen source was depleted. At the end of fermentation, final cell concentration, PHA content, and roductivity were 30.2 g/L, 44.8 % of cell dry weight, and 0.188 g/l/h, respectively.

  7. Genetic algorithm for the medium optimization of the microalga Nannochloropsis gaditana cultured to aquaculture.

    Science.gov (United States)

    Camacho-Rodríguez, J; Cerón-García, M C; Fernández-Sevilla, J M; Molina-Grima, E

    2015-02-01

    A genetic algorithm has been used to optimize the composition of the culture medium for growing the microalga Nannochloropsis gaditana, based on the nutrients composition of the commercial medium ALGAL. This strategy was carried out through the implementation of 270 experiments spread over nine generations, which allowed achieving an eicosapentaenoic acid (EPA) productivity of 17.8 m gL(-1) d(-1) in a continuous culture of N. gaditana, with an increase of 23% compared to the commercial medium. The EPA yield on nitrogen and phosphorous, 0.042 and 1.146 g(EPA) g(s)(-1), respectively, were 40% and 5-fold higher, respectively, than the values obtained with the nitrogen-optimized ALGAL medium. This improvement was obtained with the medium G-8, which also allowed reducing the requirement of several nutrients such as P, Mo, Mn in 74%, 69% and 66%, respectively, as well as the thiamine content a 46%. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Basic Techniques in Mammalian Cell Tissue Culture.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-11-01

    Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  9. Variation of Spirulina maxima biomass production in different depths of urea-used culture medium

    Science.gov (United States)

    Affan, Md-Abu; Lee, Dae-Won; Al-Harbi, Salim Marzoog; Kim, Han-Jun; Abdulwassi, Najah Ibrahim; Heo, Soo-Jin; Oh, Chulhong; Park, Heung-Sik; Ma, Chae Woo; Lee, Hyeon-Yong; Kang, Do-Hyung

    2015-01-01

    Fewer studies have assessed the outdoor cultivation of Spirulina maxima compared with S. platensis, although the protein content of S. maxima is higher than S. platensis. Spirulina growth medium requires an increased amount of NaHCO3, Na2CO3, and NaNO3, which increases the production cost. Therefore, the current study used a low-cost but high-efficiency biomass production medium (Medium M-19) after testing 33 different media. The medium depth of 25 cm (group A) was sub-divided into A1 (50% cover with a black curtain (PolyMax, 12 oz ultra-blackout), A2 (25% cover), and A3 (no cover). Similarly the medium depths of 30 and 35 cm were categorized as groups B (B1, B2, and B3) and C (C1, C2, and C3), respectively, and the effects of depth and surface light availability on growth and biomass production were assessed. The highest biomass production was 2.05 g L-1 in group A2, which was significantly higher (p Spirulina maxima died in B1 and C1 on the fifth day of culture. The biochemical composition of the biomass obtained from A2 cultures, including protein, carbohydrate, lipid, moisture, and ash, was 56.59%, 14.42%, 0.94%, 5.03%, and 23.02%, respectively. Therefore, S. maxima could be grown outdoors with the highest efficiency in urea-enriched medium at a 25-cm medium depth with 25% surface cover or uncovered. PMID:26691456

  10. Variation of Spirulina maxima biomass production in different depths of urea-used culture medium.

    Science.gov (United States)

    Affan, Md-Abu; Lee, Dae-Won; Al-Harbi, Salim Marzoog; Kim, Han-Jun; Abdulwassi, Najah Ibrahim; Heo, Soo-Jin; Oh, Chulhong; Park, Heung-Sik; Ma, Chae Woo; Lee, Hyeon-Yong; Kang, Do-Hyung

    2015-01-01

    Fewer studies have assessed the outdoor cultivation of Spirulina maxima compared with S. platensis, although the protein content of S. maxima is higher than S. platensis. Spirulina growth medium requires an increased amount of NaHCO3, Na2CO3, and NaNO3, which increases the production cost. Therefore, the current study used a low-cost but high-efficiency biomass production medium (Medium M-19) after testing 33 different media. The medium depth of 25 cm (group A) was sub-divided into A1 (50% cover with a black curtain (PolyMax, 12 oz ultra-blackout), A2 (25% cover), and A3 (no cover). Similarly the medium depths of 30 and 35 cm were categorized as groups B (B1, B2, and B3) and C (C1, C2, and C3), respectively, and the effects of depth and surface light availability on growth and biomass production were assessed. The highest biomass production was 2.05 g L-1 in group A2, which was significantly higher (p Spirulina maxima died in B1 and C1 on the fifth day of culture. The biochemical composition of the biomass obtained from A2 cultures, including protein, carbohydrate, lipid, moisture, and ash, was 56.59%, 14.42%, 0.94%, 5.03%, and 23.02%, respectively. Therefore, S. maxima could be grown outdoors with the highest efficiency in urea-enriched medium at a 25-cm medium depth with 25% surface cover or uncovered.

  11. Variation of Spirulina maxima biomass production in different depths of urea-used culture medium

    Directory of Open Access Journals (Sweden)

    Md-Abu Affan

    2015-01-01

    Full Text Available Fewer studies have assessed the outdoor cultivation of Spirulina maxima compared with S. platensis, although the protein content of S. maxima is higher than S. platensis. Spirulina growth medium requires an increased amount of NaHCO3, Na2CO3, and NaNO3, which increases the production cost. Therefore, the current study used a low-cost but high-efficiency biomass production medium (Medium M-19 after testing 33 different media. The medium depth of 25 cm (group A was sub-divided into A1 (50% cover with a black curtain (PolyMax, 12 oz ultra-blackout, A2 (25% cover, and A3 (no cover. Similarly the medium depths of 30 and 35 cm were categorized as groups B (B1, B2, and B3 and C (C1, C2, and C3, respectively, and the effects of depth and surface light availability on growth and biomass production were assessed. The highest biomass production was 2.05 g L-1 in group A2, which was significantly higher (p < 0.05 than that in all other groups and sub-groups. Spirulina maxima died in B1 and C1 on the fifth day of culture. The biochemical composition of the biomass obtained from A2 cultures, including protein, carbohydrate, lipid, moisture, and ash, was 56.59%, 14.42%, 0.94%, 5.03%, and 23.02%, respectively. Therefore, S. maxima could be grown outdoors with the highest efficiency in urea-enriched medium at a 25-cm medium depth with 25% surface cover or uncovered.

  12. Culture of Ovine IVM/IVF Zygotes in Isolated Mouse Oviduct: Effect of Basal Medium

    OpenAIRE

    Farahavar, Abbas; Shirazi, Abolfazl; Kohram, Hamid; Shahneh, Ahmad Zareh; Sarvari, Ali; Naderi, Mohammad Mehdi; Boroujeni, Sara Borjian; Zhandi, Mehdi

    2013-01-01

    Background The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. Methods The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated ovi...

  13. In vitro culture medium (IVC) supplementation with sericin improves developmental competence of ovine zygotes.

    Science.gov (United States)

    Aghaz, Faranak; Hajarian, Hadi; KaramiShabankareh, Hamed

    2016-03-01

    This study was carried out to investigate the effects of supplementation of potassium simplex optimized medium (KSOM-aa) with various sericin concentrations (0, 0.1, 0.5, 1 and 2.5%) on ovine zygotes. The results indicate that the supplementation of oocyte in vitro culture medium with optimal concentration of sericin (0.1 and 0.5%) may have beneficial effects on developmental competence of in vitro-derived ovine embryos. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  14. Plasma-activated medium (PAM) kills human cancer-initiating cells.

    Science.gov (United States)

    Ikeda, Jun-Ichiro; Tanaka, Hiromasa; Ishikawa, Kenji; Sakakita, Hajime; Ikehara, Yuzuru; Hori, Masaru

    2018-01-01

    Medical non-thermal plasma (NTP) treatments for various types of cancers have been reported. Cells with tumorigenic potential (cancer-initiating cells; CICs) are few in number in many types of tumors. CICs efficiently eliminate anti-cancer chemicals and exhibit high-level aldehyde dehydrogenase (ALDH) activity. We previously examined the effects of direct irradiation via NTP on cancer cells; even though we targeted CICs expressing high levels of ALDH, such treatment affected both non-CICs and CICs. Recent studies have shown that plasma-activated medium (PAM) (culture medium irradiated by NTP) selectively induces apoptotic death of cancer but not normal cells. Therefore, we explored the anti-cancer effects of PAM on CICs among endometrioid carcinoma and gastric cancer cells. PAM reduced the viability of cells expressing both low and high levels of ALDH. Combined PAM/cisplatin appeared to kill cancer cells more efficiently than did PAM or cisplatin alone. In a mouse tumor xenograft model, PAM exerted an anti-cancer effect on CICs. Thus, our results suggest that PAM effectively kills both non-CICs and CICs, as does NTP. Therefore, PAM may be a useful new anti-cancer therapy, targeting various cancer cells including CICs. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  15. Atrazine and its metabolites degradation in mineral salts medium and soil using an enrichment culture.

    Science.gov (United States)

    Kumar, Anup; Singh, Neera

    2016-03-01

    An atrazine-degrading enrichment culture was used to study degradation of atrazine metabolites viz. hydroxyatrazine, deethylatrazine, and deisopropylatrazine in mineral salts medium. Results suggested that the enrichment culture was able to degrade only hydroxyatrazine, and it was used as the sole source of carbon and nitrogen. Hydroxyatrazine degradation slowed down when sucrose and/or ammonium hydrogen phosphate were supplemented as the additional sources of carbon and nitrogen, respectively. The enrichment culture could degrade high concentrations of atrazine (up to 110 μg/mL) in mineral salts medium, and neutral pH was optimum for atrazine degradation. Further, except in an acidic soil, enrichment culture was able to degrade atrazine in three soil types having different physico-chemical properties. Raising the pH of acidic soil to neutral or alkaline enabled the enrichment culture to degrade atrazine suggesting that acidic pH inhibited atrazine-degrading ability. The study suggested that the enrichment culture can be successfully utilized to achieve complete degradation of atrazine and its persistent metabolite hydroxyatrazine in the contaminated soil and water.

  16. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  17. Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

    Directory of Open Access Journals (Sweden)

    Carlos Andrés Zárate-Chaves

    2013-01-01

    Full Text Available The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

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

    Science.gov (United States)

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

    2009-01-01

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

  19. Raman microspectroscopy: a noninvasive analysis tool for monitoring of collagen-containing extracellular matrix formation in a medium-throughput culture system.

    Science.gov (United States)

    Kunstar, Aliz; Otto, Cees; Karperien, Marcel; van Blitterswijk, Clemens; van Apeldoorn, Aart

    2011-07-01

    The three-dimensional environment is known to play an important role in promoting cell-matrix interactions. We have investigated the possibility of using Raman microspectroscopy--which has the great advantage of noninvasive sensing--for in vitro monitoring of extracellular matrix (ECM) formation in a medium-throughput pellet (3D) culture system with soft-litography, agarose-microwell arrays. Chondrocytes were seeded in the agarose microwells in basic or chondrocyte medium. After 3, 7, and 14 days of culture, samples were analyzed for ECM formation by Raman microspectroscopy, histology, and immunofluorescence. ECM formation in the chondrocyte medium-cultured samples was detected by histology and immunofluorescence, and also noninvasively by Raman microspectroscopy. The Raman band of collagen found at 937 cm(-1) can be used as a Raman marker for collagen-containing ECM formation over time in the chondrocyte pellets. This culture system can be implemented as a medium-throughput platform for Raman applications and screening microtissue formation, since with these agarose-microwell arrays relatively large numbers of cell pellets could be screened in a short time in situ, without having to transfer the pellets onto microscopic slides. Moreover, in this manner the culture system is suitable for long-term, real-time live-cell measurements.

  20. Ability of a Lactobacillus rhamnosus strain cultured in milk whey based medium to bind aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Fernanda Bovo

    2014-09-01

    Full Text Available This study aimed to compare Lactobacillus rhamnosus growth in MRS (de Man, Rogosa and Sharpe broth and a culture medium containing milk whey (MMW and to evaluate aflatoxin B1 (AFB1 adsorption capacity by bacterial cells produced in both culture media. L. rhamnosus cells were cultivated in MRS broth and MMW (37 °C, 24 hours, and bacterial cell concentration was determined spectrophotometrically at 600 nm. AFB1 (1 µg/ml adsorption assays were conducted using 1 x 10(10 non-viable L. rhamnosus cells (121 °C, 15 minutes at pHs 3.0 and 6.0 and contact time of 60 minutes. AFB1 quantification was performed by High Performance Liquid Chromatography. Bacterial cell concentration in MMW was higher (9.84 log CFU/ml than that in MRS broth (9.63 log CFU/ml. There were no significant differences between AFB1 binding results at the same pH value (3.0 or 6.0 for the cells cultivated in MRS broth (46.0% and 35.8%, respectively and in MMW (43.7% and 25.8%, respectively, showing that MMW can adequately replace the MRS broth. Therefore, it can be concluded that the use of L. rhamnosus cells cultivated in MMW offers advantages such as reduction in large scale production costs, improvement of environmental sustainability, and being a practicable alternative for decontamination of food products susceptible to aflatoxin contamination.

  1. Growth of melanocytes in human epidermal cell cultures

    International Nuclear Information System (INIS)

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C.

    1990-01-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient

  2. IVF culture medium affects post-natal weight in humans during the first 2 years of life

    NARCIS (Netherlands)

    Kleijkers, S.H.; Montfoort, A.P. van; Smits, L.J.; Viechtbauer, W.; Roseboom, T.J.; Nelissen, E.C.; Coonen, E.; Derhaag, J.G.; Bastings, L.; Schreurs, I.E.; Evers, J.L.H.; Dumoulin, J.C.

    2014-01-01

    STUDY QUESTION: Is post-natal growth during the first 2 years of life in IVF singletons affected by type of medium used for culturing human embryos during an IVF treatment? SUMMARY ANSWER: The in vitro culture of human embryos in medium from Cook resulted in singletons with a lower weight during the

  3. IVF culture medium affects post-natal weight in humans during the first 2 years of life

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; van Montfoort, Aafke P. A.; Smits, Luc J. M.; Viechtbauer, Wolfgang; Roseboom, Tessa J.; Nelissen, Ewka C. M.; Coonen, Edith; Derhaag, Josien G.; Bastings, Lobke; Schreurs, Inge E. L.; Evers, Johannes L. H.; Dumoulin, John C. M.

    2014-01-01

    Is post-natal growth during the first 2 years of life in IVF singletons affected by type of medium used for culturing human embryos during an IVF treatment? The in vitro culture of human embryos in medium from Cook resulted in singletons with a lower weight during the first 2 years of life compared

  4. Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

    Science.gov (United States)

    Kaneko, Ai; Sankai, Yoshiyuki

    2014-01-01

    The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

  5. Optimization of culturing condition and medium composition for the production of alginate lyase by a marine Vibrio sp. YKW-34

    Science.gov (United States)

    Fu, Xiaoting; Lin, Hong; Kim, Sang Moo

    2008-02-01

    Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL-1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25°C. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

  6. Cultivar, explant type and culture medium influencing embryogenesis and organogenegenesis in Anthurium spp.

    Directory of Open Access Journals (Sweden)

    Yaowaphan Sontikun

    2006-07-01

    Full Text Available The effect of cultivars, culture media, explant type and wounding on culture response of anthurium were studied. Valantino gave the highest callus formation (83.73% significantly different from Sonat (78.73% and Plew Thien Phuket (45.66%. Valantino and Plew Thien Phuket gave meristematic nodular callus (MNC whereas Sonat produced embryogenic-like callus (ELC. Modified Murashige and Skoog (MMS medium gave the highest callus formation from both leaf (86.6% and node (100%. Callus obtained in Nitsch and Nitsch (NN and MMS was MNC while woody plant (WPM medium provided ELC. For explant types, internode gave the highest callus formation (72.63%. Nodal and internodal explant gave ELC while the leaf explant yielded MNC. Wounding leaf blades tended to promote more MNC.

  7. An improved selective culture medium enhances the isolation of Burkholderia pseudomallei from contaminated specimens.

    Science.gov (United States)

    Goodyear, Andrew; Strange, Linda; Rholl, Drew A; Silisouk, Joy; Dance, David A B; Schweizer, Herbert P; Dow, Steven

    2013-11-01

    Burkholderia pseudomallei is a Gram-negative environmental bacterium found in tropical climates that causes melioidosis. Culture remains the diagnostic gold standard, but isolation of B. pseudomallei from heavily contaminated sites, such as fecal specimens, can be difficult. We recently reported that B. pseudomallei is capable of infecting the gastrointestinal tract of mice and suggested that the same may be true in humans. Thus, there is a strong need for new culture techniques to allow for efficient detection of B. pseudomallei in fecal and other specimens. We found that the addition of norfloxacin, ampicillin, and polymyxin B to Ashdown's medium (NAP-A) resulted in increased specificity without affecting the growth of 25 B. pseudomallei strains. Furthermore, recovery of B. pseudomallei from human clinical specimens was not affected by the three additional antibiotics. Therefore, we conclude that NAP-A medium provides a new tool for more sensitive isolation of B. pseudomallei from heavily contaminated sites.

  8. Culturing Selenastrum capricornutum (Chlorophyta) in a synthetic algal nutrient medium with defined mineral particulates

    Science.gov (United States)

    Kuwabara, J.S.; Davis, J.A.; Chang, Cecily C.Y.

    1985-01-01

    Algal nutrient studies in chemically-defined media typically employ a synthetic chelator to prevent iron hydroxide precipitation. Micronutrient-particulate interactions may, however, significantly affect chemical speciation and hence biovailability of these nutrients in natural waters. A technique is described by which Selenastrum capricornutum Printz (Chlorophyta) may be cultured in a medium where trace metal speciation (except iron) is controlled, not by organic chelation, but by sorption onto titanium dioxide. Application of this culturing protocol in conjunction with results from sorption studies of nutrient ions on mineral particles provides a means of studying biological impacts of sorptive processes in aquatic environments. ?? 1985 Dr W. Junk Publishers.

  9. The Effect of Culture Medium on Metabolic and Antibacterial Activities of Probiotic Bacteria

    Directory of Open Access Journals (Sweden)

    Mirdavoudi F

    2012-01-01

    Full Text Available Background and Objectives: Probiotic bacteria is added directly to food components and it has beneficial effect on function and the health of organisms. The bifidogenic factors enter the colon where they contribute to an increase lactic acid bacteria population including Lactobacilli and Bifidobacteria and they inhibit enteric pathogenic bacterial growth. The aim of this study is to investigate the effect of culture medium on metabolic and antibacterial of probiotic bacteria.Methods: In this study, the probiotics bacterial and intestine pathogenic are to be used. Lactobacilli and Bifidobacterium were identified by plating samples on MRS medium, Gram Staining and standard biochemical methods. The effect of antagonistic probiotics was investigated in the presence of growth factor in the method well diffusion Ager on the Shigella flexneri (PTCC 1234, Escherichia coli (PTCC 1552, Salmonella typhi ( PTCC 1609 and the culture medium pH was measured.Results: The probiotics bacterial growth in MRS and lactose1%, sorbitol, raffinose, riboflavin were shown the effect antibacterial. The results of the study show the most antagonistic activity in commercial strain Lactobacillus acidophilus on Shigella flexneri and lower activity was in Lactobacillus casei (PTCC 1608, and Salmonella typhimurium (PTCC 1609, and also in Bbifidobacterium bifidum, it showed the most decrease pH value.Conclusion: According to the result of the study, adding growth factors to MRS medium base and lactose 1%, probiotic growth was increased and which also increased antagonistic activity.

  10. Adapting the Medium: Dynamics of Intermedial Adaptation in Contemporary Japanese Popular Visual Culture

    Directory of Open Access Journals (Sweden)

    Pusztai Beáta

    2015-08-01

    Full Text Available With respect to adaptation studies, contemporary Japanese popular culture signifies a unique case, as different types of media (be those textual, auditive, visual or audio-visual are tightly intertwined through the “recycling” of successful characters and stories. As a result, a neatly woven net of intermedial adaptations has been formed - the core of this complex system being the manga-anime-live-action film “adaptational triangle.” On the one hand, the paper addresses the interplay of the various factors by which the very existence of this network is made possible, such as the distinctive cultural attitude to “originality,” the structure of the comics, animation and film industries, and finally, the role of fictitious genealogies of both traditional and contemporary media in the negotiation of national identity. On the other hand, the essay also considers some of the most significant thematic, narrative, and stylistic effects this close interconnectedness has on the individual medium. Special attention is being paid to the nascent trend of merging the adaptive medium with that of the original story (viewing adaptation as integration, apparent in contemporary manga-based live- action comedies, as the extreme case of intermedial adaptation. That is, when the aim of the adaptational process is no longer the transposition of the story but the adaptation (i.e. the incorporation of the medium itself- elevating certain medium-specific devices into transmedial phenomena.

  11. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

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

  12. Evaluation of the addition of glycerol to Cupriavidus necator culture medium over Poly(3-hydroxybutyrate) production

    OpenAIRE

    Apati, Giannini Paszinick; Kelbert, Maikon; Sombrio, Bruna Regina; Schneider, Andrea Lima dos Santos; Garcia, Michele Cristina Formolo; Furigo Junior, Agenor; Pezzin, Ana Paula Testa

    2018-01-01

    ABSTRACT Glycerol was used as a source of additional carbon in the production of Poly(3-hydroxybutyrate) (P(3HB)). The inverted sugar and glycerol concentrations and the temperature of the Cupriavidus necator culture medium were evaluated using a Central Composite Rotational Design (CCRD). The results showed that the increase in temperature and sugar concentration led to an increase in production and P(3HB) accumulation and when 15 g L-1 of glycerol was added better results were obtained, how...

  13. Unrestricted somatic stem cells from human umbilical cord blood grow in serum-free medium as spheres

    Directory of Open Access Journals (Sweden)

    Ang Haozhi

    2009-12-01

    Full Text Available Abstract Background Human umbilical cord blood-derived unrestricted somatic stem cells (USSCs, which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. A major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. In this study, we investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGRO™, PSM and USSC growth mediumACF. Our findings demonstrate that USSCs do not grow in HEScGRO™ or PSM, but we were able to isolate, proliferate and maintain multipotency of three USSC lines in USSC growth mediumACF. Results For the first one to three passages, cells grown in USSC growth mediumACF proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. Upon dissociation of spheres, cells continue to grow in suspension and form new spheres. Dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support (fibronectin or gelatin, or in medium containing fetal calf serum. Analysis of markers associated with pluripotency (Oct4 and Sox2 and differentiation (FoxA2, Brachyury, Goosecoid, Nestin, Pax6, Gata6 and Cytokeratin 8 confirms that cells in the spheres maintain their gene expression profile. The cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages. Conclusions These data suggest that USSC growth mediumACF maintains USSCs in an undifferentiated state and supports growth in suspension. This is the first demonstration that USSCs can grow in a serum- and animal component-free medium and that USSCs can form spheres.

  14. Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

    OpenAIRE

    van Luyn, M.J.A.; van Wachem, P.B.; Nieuwenhuis, P.; Olde-Damink, L.; ten Hoopen, Hermina W.M.; Feijen, Jan

    1991-01-01

    The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic products. The agar-overlay test is a short term semi-solid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell free zones. The aim of this ...

  15. METHYLCELLULOSE CELL-CULTURE AS A NEW CYTOTOXICITY TEST SYSTEM FOR BIOMATERIALS

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; DAMINK, LO; TENHOOPEN, H; FEIJEN, J

    The cytotoxicity of biomaterials can be tested in vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released

  16. Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

    NARCIS (Netherlands)

    van Luyn, M.J.A.; van Wachem, P.B.; Nieuwenhuis, P.; Olde-Damink, L.; ten Hoopen, Hermina W.M.; Feijen, Jan

    1991-01-01

    The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic

  17. Effect of explant density and medium culture volumes on cassava micropropagation in Temporal Immersion System

    Directory of Open Access Journals (Sweden)

    Milagros Basail

    2003-04-01

    Full Text Available Due to the need of producing high quality planting material available to cassava growers, it has been necessary to look for alternatives in order to increase the efficiancy of in vitro propagation methods and their automation, such as the use of the Temporal Immersion Systems (RITA®. This work was carried out to increase the multiplication coefficient for cassava mass propagation through out Temporal Immersion Systems. The clone ‘CMC-40’ was used. Different medium volumes per explant, and material density per unit at a given Immersion frequency were tested. The highest results were obtained in the 2.8 multiplication coefficient with 20 ml culture medium volume and 3.2 using a density of 40 explants/flask. When the Temporal Immersion System is used with these results, a more efficient method for cassava micropropagation is established and also higher quality vitroplants for the rooting stage and further acclimatization in field conditions are produced. Key Words: Tissue Culture, liquid culture medium, Manihot esculenta Crantz

  18. Growth performances and changes of macronutrient ion concentrations in the culture medium when Euglena gracilis was cultured with nitrified digestate.

    Science.gov (United States)

    Takemura, Kenji; Endo, Ryosuke; Shibuya, Toshio; Kitaya, Yoshiaki

    2017-09-01

    We investigated the possibility of using Euglena gracilis to convert digestate from methane fermentation of organic wastes into a medium for soilless crop culture. The growth of E. gracilis cultured with aqueous solutions containing filtrate of raw digestate at 1-30% (v/v) and nitrified digestate at 10-100% (v/v) was examined. Concentrations of plant macronutrient ions in nitrified digestate before and after culturing E. gracilis were also examined. Specific growth rates in aqueous solutions containing filtrate of raw digestate at 1-10% and nitrified digestate at 10-100% showed no significant differences, respectively (0.781 ± 0.031 d -1 and 0.925 ± 0.033 d -1 , mean ± standard error). The rates in the filtrate of nitrified digestate were significantly higher than those in the filtrate of raw digestate. Moreover, there were no significant differences between the concentrations of plant macronutrient ions other than [Formula: see text] in the filtrate of nitrified digestate before and after culturing E. gracilis. The concentration of [Formula: see text] decreased significantly by 10.5% of the initial concentration. As a result, the constituent ratio of plant macronutrient ions other than magnesium in the solution after culturing E. gracilis was similar to that in a standard nutrient solution for soilless culture.

  19. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype.

    Science.gov (United States)

    Huang, Chen; Zhang, Jing; Ao, Mingxin; Li, Ying; Zhang, Chun; Xu, Yonggen; Li, Xuemin; Wang, Wei

    2012-02-01

    Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction. Copyright © 2011 Wiley Periodicals, Inc.

  20. Optimization of culture conditions and medium composition for the marine algicidal bacterium Alteromonas sp. DH46 by uniform design

    Science.gov (United States)

    Lin, Jing; Zheng, Wei; Tian, Yun; Wang, Guizhong; Zheng, Tianling

    2013-09-01

    Harmful algal blooms (HABs) have led to extensive ecological and environmental issues and huge economic losses. Various HAB control techniques have been developed, and biological methods have been paid more attention. Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner, and kill or damage the algal cells. A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp. The culture conditions were optimized using a single-factor test method. Factors including carbon source, nitrogen source, temperature, initial pH value, rotational speed and salinity were studied. The results showed that the cultivation of the bacteria at 28°C and 180 r min-1 with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46. The optimal medium composition for strain DH46 was determined by means of uniform design experimentation, and the most important components influencing the cell density were tryptone, yeast extract, soluble starch, NaNO3 and MgSO4. When the following culture medium was used (tryptone 14.0g, yeast extract 1.63g, soluble starch 5.0 g, NaNO3 1.6 g, MgSO4 2.3 g in 1L), the largest bacterial dry weight (7.36 g L-1) was obtained, which was an enhancement of 107% compared to the initial medium; and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.

  1. Hypoxic contraction of cultured pulmonary vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Murray, T.R.; Chen, L.; Marshall, B.E.; Macarak, E.J.

    1990-01-01

    The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells

  2. Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Mee-Hae Kim

    2017-10-01

    Full Text Available In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL, which can be set to 37 °C and 5% CO2, and uses a gas-permeable resin (poly- dimethylsiloxane as the vessel wall. RPE cells were seeded at 5.0 × 104 cells/cm2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

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

    International Nuclear Information System (INIS)

    Sajid, Z.A.

    2016-01-01

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

  4. Organizational Culture and Open Innovation Performance in Small and Medium-sized Enterprises (SMEs in Poland

    Directory of Open Access Journals (Sweden)

    Mazur Jolanta

    2016-09-01

    Full Text Available This study investigates the links between organizational culture, the use of open innovation sources and the performance of SMEs. The main hypothesis of the study is that a special type of organizational culture (termed innovative culture, which fosters creativity, learning and inter-employee cooperation – will correspond with a greater scope of open innovation sources and higher levels of innovative, operational and financial performance. The study was based on a representative CATI survey of 473 SMEs operating in manufacturing and services industries in Poland. Our statistical analysis relied on building and testing structural equation model with the AMOS software. The findings confirmed a positive association between innovative culture and the scope of open sources of innovation. However, innovative culture had no direct effect on the percentage of sales from new and modified products, which is often used as a metric of innovativeness, but did show a positive influence on an index of operational performance and ROI. Such statistical patterns suggest that fostering innovative culture is beneficial to a company, though probably not through an increased number of product innovations, but rather via process, administrative and marketing innovations, as well as other gains in efficiency attained due to more streamlined employee cooperation and knowledge exchange. The study adds to the existing body of knowledge in management science by providing a better understanding of mechanisms underlying innovative culture’s impacts on open innovation practices and metrics of operational and financial performance in the context of small and medium enterprises.

  5. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF : a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten - Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  6. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, S.H.; Mantikou, E.; Slappendel, E.; Consten, D.; Echten-Arends, J. van; Wetzels, A.M.M.; Wely, M. van; Smits, L.J.; Montfoort, A.P. van; Repping, S.; Dumoulin, J.C.; Mastenbroek, S.

    2016-01-01

    STUDY QUESTION: Does embryo culture medium influence pregnancy and perinatal outcome in IVF? SUMMARY ANSWER: Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. WHAT IS KNOWN ALREADY: A wide variety of culture media for human preimplantation embryos in

  7. Influence of embryo culture medium (G5 and HTF) on pregnancy and perinatal outcome after IVF: a multicenter RCT

    NARCIS (Netherlands)

    Kleijkers, Sander H. M.; Mantikou, Eleni; Slappendel, Els; Consten, Dimitri; van Echten-Arends, Jannie; Wetzels, Alex M.; van Wely, Madelon; Smits, Luc J. M.; van Montfoort, Aafke P. A.; Repping, Sjoerd; Dumoulin, John C. M.; Mastenbroek, Sebastiaan

    2016-01-01

    Does embryo culture medium influence pregnancy and perinatal outcome in IVF? Embryo culture media used in IVF affect treatment efficacy and the birthweight of newborns. A wide variety of culture media for human preimplantation embryos in IVF/ICSI treatments currently exists. It is unknown which

  8. Designing media for animal cell culture: CHO cells, the industrial standard.

    Science.gov (United States)

    Landauer, Karlheinz

    2014-01-01

    The success of culturing CHO cells solely depends on functionality of the used media. Cell culture technology is more than 50 years old, and the knowledge of cell requirements increased steadily. In the beginning, animal-sourced components were the key to growth. Nowadays state-of-the-art media do not contain any animal or naturally sourced components. The compositions are based on scientific awareness of the needs of the cells. The result is high lot-to-lot consistency and high performance.In this book section, a method for the development of a synthetic, animal component-free medium is described. The composition is based on public available formulations and information based on the work of many scientists printed in numerous papers and manuscripts. The method shall help beginners to design their own medium, although some knowledge of biochemistry and animal cells is still required.

  9. Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

    Science.gov (United States)

    Conceição, M M; Tonso, A; Freitas, C B; Pereira, C A

    2007-11-07

    Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected

  10. Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.

    Science.gov (United States)

    Tinoco, Raunel; Acevedo, Abisaí; Galindo, Enrique; Serrano-Carreón, Leobardo

    2011-04-01

    Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2⁶⁻² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.

  11. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco.

  12. Timescale of silver nanoparticle transformation in neural cell cultures impacts measured cell response

    Energy Technology Data Exchange (ETDEWEB)

    Hume, Stephanie L.; Chiaramonti, Ann N.; Rice, Katherine P.; Schwindt, Rani K. [National Institute of Standards and Technology (NIST), Applied Chemicals and Materials Division (United States); MacCuspie, Robert I. [National Institute of Standards and Technology (NIST), Materials Measurement Science Division (United States); Jeerage, Kavita M., E-mail: jeerage@boulder.nist.gov [National Institute of Standards and Technology (NIST), Applied Chemicals and Materials Division (United States)

    2015-07-15

    Both serum protein concentration and ionic strength are important factors in nanoparticle transformation within cell culture environments. However, silver nanoparticles are not routinely tracked at their working concentration in the specific medium used for in vitro toxicology studies. Here we evaluated the transformation of electrostatically stabilized citrate nanoparticles (C-AgNPs) and sterically stabilized polyvinylpyrrolidone nanoparticles (PVP-AgNPs) in a low-serum (∼ 0.2 mg/mL bovine serum albumin) culture medium, while measuring the response of rat cortex neural progenitor cells, which differentiate in this culture environment. After 24 h, silver nanoparticles at concentrations up to 10 µg/mL did not affect adenosine triphosphate levels, whereas silver ions decreased adenosine triphosphate levels at concentrations of 1.1 µg/mL or higher. After 240 h, both silver nanoparticles, as well as silver ion, unambiguously decreased adenosine triphosphate levels at concentrations of 1 and 1.1 µg/mL, respectively, suggesting particle dissolution. Particle transformation was investigated in 1:10 diluted, 1:2 diluted, or undiluted differentiation medium, all having an identical protein concentration, to separate the effect of serum protein stabilization from ionic strength destabilization. Transmission electron microscopy images indicated that particles in 1:10 medium were not surrounded by proteins, whereas particles became clustered within a non-crystalline protein matrix after 24 h in 1:2 medium and at 0 h in undiluted medium. Despite evidence for a protein corona, particles were rapidly destabilized by high ionic strength media. Polyvinylpyrrolidone increased the stability of singly dispersed particles compared to citrate ligands; however, differences were negligible after 4 h in 1:2 medium or after 1 h in undiluted medium. Thus low-serum culture environments do not provide sufficient colloidal stability for long-term toxicology studies with citrate

  13. Timescale of silver nanoparticle transformation in neural cell cultures impacts measured cell response

    International Nuclear Information System (INIS)

    Hume, Stephanie L.; Chiaramonti, Ann N.; Rice, Katherine P.; Schwindt, Rani K.; MacCuspie, Robert I.; Jeerage, Kavita M.

    2015-01-01

    Both serum protein concentration and ionic strength are important factors in nanoparticle transformation within cell culture environments. However, silver nanoparticles are not routinely tracked at their working concentration in the specific medium used for in vitro toxicology studies. Here we evaluated the transformation of electrostatically stabilized citrate nanoparticles (C-AgNPs) and sterically stabilized polyvinylpyrrolidone nanoparticles (PVP-AgNPs) in a low-serum (∼ 0.2 mg/mL bovine serum albumin) culture medium, while measuring the response of rat cortex neural progenitor cells, which differentiate in this culture environment. After 24 h, silver nanoparticles at concentrations up to 10 µg/mL did not affect adenosine triphosphate levels, whereas silver ions decreased adenosine triphosphate levels at concentrations of 1.1 µg/mL or higher. After 240 h, both silver nanoparticles, as well as silver ion, unambiguously decreased adenosine triphosphate levels at concentrations of 1 and 1.1 µg/mL, respectively, suggesting particle dissolution. Particle transformation was investigated in 1:10 diluted, 1:2 diluted, or undiluted differentiation medium, all having an identical protein concentration, to separate the effect of serum protein stabilization from ionic strength destabilization. Transmission electron microscopy images indicated that particles in 1:10 medium were not surrounded by proteins, whereas particles became clustered within a non-crystalline protein matrix after 24 h in 1:2 medium and at 0 h in undiluted medium. Despite evidence for a protein corona, particles were rapidly destabilized by high ionic strength media. Polyvinylpyrrolidone increased the stability of singly dispersed particles compared to citrate ligands; however, differences were negligible after 4 h in 1:2 medium or after 1 h in undiluted medium. Thus low-serum culture environments do not provide sufficient colloidal stability for long-term toxicology studies with citrate

  14. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  15. Extension of the Lifespan of Cultured Normal Human Diploid Cells by Vitamin E

    Science.gov (United States)

    Packer, Lester; Smith, James R.

    1974-01-01

    Inclusion of vitamin E (DL-α-tocopherol) in the culture medium for human diploid cells greatly prolongs their in vitro lifespan. The addition of 100 μg of DL-α-tocopherol per ml of medium has allowed us to culture WI-38 cells for more than 100 population doublings to date. (These cells normally have an in vitro lifespan of 50 ± 10 population doublings.) Cells at the 100th population doubling have a normal diploid karyotype, appear to behave in all other respects like young WI-38 cells, and are still actively dividing. We interpret this result as support for the free radical theory of aging. Images PMID:4531015

  16. Acute toxicity bioassays using Daphnia magna Straus (Cladocera, Daphniidae maintained in a modified culture medium

    Directory of Open Access Journals (Sweden)

    Mónica Núñez

    2013-05-01

    Full Text Available Daphnia magna is a test organism used in ecotoxicological assays of freshwater; however, traditional culture systems for this organism could result expensive, for that the aim of this research was to developed a new economic culture medium. With this purpose, 10 strains of D. magna were isolated, their population development was evaluated by total count of organisms and pregnant females using 3 different culture media: (A alfalfa juice, (B solved yeast and (C a mixture of alfalfa juice plus solved yeast. Successful development of 4 strains was observed in the A medium, but the same strains failed to survive in the B and the C media. The 24h and 48h EC50 average values in acute ecotoxicological assays with potassium dicromate were 0,4045 mg/L ± 0,0389 and 0,1857 mg/L ± 0,0072 respectively. Also, acute ecotoxicological assays with these 4 strains were performed using potassium cyanide, which is a toxic reactive frequently used in mining operations. In this case 24h EC50 value was 1,5388 mg/L ± 0,1146 and 48h EC50 values were 0,6359 mg/L ± 0,0516. 48h EC50 values were lower than the cyanide permissible effluent values established by the Energy and Mining Authority.

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

    Directory of Open Access Journals (Sweden)

    Aleksandra Seta-Koselska

    2018-01-01

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

  18. Resource efficiency and culture--workplace training for small and medium-sized enterprises.

    Science.gov (United States)

    Bliesner, Anna; Liedtke, Christa; Rohn, Holger

    2014-05-15

    Although there are already some qualification offers available for enterprises to support resource efficiency innovations, the high potentials that can be identified especially for small and medium sized enterprises (SMEs) have not been activated until now. As successful change lies in the hands of humans, the main aim of vocational education has to be the promotion of organisational and cultural changes in the enterprises. As there is already a small but increasing number of enterprises that perform very well in resource efficiency innovations one question arises: What are typical characteristics of those enterprises? Leaning on a good-practice approach, the project "ResourceCulture" is going to prove or falsify the hypothesis that enterprises being successful with resource efficiency innovations have a specific culture of trust, which substantially contributes to innovation processes, or even initially enables them. Detailed empirical field research will light up which correlations between resource efficiency, innovation and cultures of trust can be found and will offer important aspects for the improvement of management instruments and qualification concepts for workplace training. The project seizes qualification needs that were likewise mentioned by enterprises and consultants, regarding the implementation of resource efficiency. This article - based on first empirical field research results - derives preliminary indications for the design of the qualification module for the target groups resource efficiency consultants and managers. On this basis and in order to implement "ResourceCulture" conceptual and methodological starting points for workplace training are outlined. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Growth and phenotypic characteristics of human nevus cells in culture.

    Science.gov (United States)

    Mancianti, M L; Herlyn, M; Weil, D; Jambrosic, J; Rodeck, U; Becker, D; Diamond, L; Clark, W H; Koprowski, H

    1988-02-01

    Nevus cells were isolated from the three cutaneous components, epidermis, basal layer, and dermis, of nonmalignant pigmented lesions and were cultured separately in the presence or absence of the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate in medium that supports the rapid proliferation of melanocytic cells. The separation procedure used provided cultures that were essentially free from normal melanocytes (dermis) or fibroblasts (epidermis). In short term culture, nevus cells of all skin compartments expressed markers associated with differentiated melanocytes, such as presence of premelanosomes and melanosomes and elevated tyrosinase levels. Nevus cells also expressed melanoma-associated antigens, such as NGF-receptor, transferrin-related p97, proteoglycan, and HLA-DR as detected with monoclonal antibodies. After several subpassages, cells showed a decreased expression of melanoma-associated antigens, decreased tyrrosinase levels, and melanosomes could no longer be detected. Morphologically, these cells were similar to fibroblasts. The disappearance of melanoma-associated cell surface antigens was concomitant with the appearance of a melanocyte-associated 145 kd protein that might serve as a marker of fibroblast-like differentiation in nevus cells and normal melanocytes. Nevus cell cultures grown in the presence of 12-0-tetradecanoyl phorbol-13-acetate maintained a stable differentiated phenotype throughout their lifespan. As reported earlier, nevus cells in culture, irrespective of the presence or absence of 12-0-tetradecanoyl phorbol-13-acetate, have a finite lifespan in vitro, grow anchorage-independent in soft agar, but do not form tumors when xenografted to nude mice. These studies demonstrate that nevus cells isolated from the epidermal, basal layer, and dermal components of lesional skin can serve as models to characterize the initial steps of tumor progression in a human cell system.

  20. Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium

    International Nuclear Information System (INIS)

    Apel, Anja; Groth, Ariane; Schlesinger, Sabine; Bruns, Helge; Schemmer, Peter; Buechler, Markus W.; Herr, Ingrid

    2009-01-01

    Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells

  1. Animal-cell culture media: History, characteristics, and current issues.

    Science.gov (United States)

    Yao, Tatsuma; Asayama, Yuta

    2017-04-01

    Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

  2. Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells.

    Science.gov (United States)

    Kadekar, Darshana; Rangole, Sonal; Kale, Vaijayanti; Limaye, Lalita

    2016-01-01

    The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells. CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them. Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

  3. Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells.

    Directory of Open Access Journals (Sweden)

    Darshana Kadekar

    Full Text Available The limited cell dose in umbilical cord blood (UCB necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS. Conditioned medium from mesenchymal stem cells (MSCs-CM has been shown to alleviate the oxidative stress during wound healing, Alzheimer's disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells.CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM. UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them.Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability.

  4. Cultured stem cells are sensitive to gravity changes

    Science.gov (United States)

    Buravkova, L. B.; Romanov, Yu. A.; Konstantinova, N. A.; Buravkov, S. V.; Gershovich, Yu. G.; Grivennikov, I. A.

    2008-09-01

    Stem and precursor cells play an important role in development and regeneration. The state of these cells is regulated by biochemical substances, mechanical stimuli and cellular interactions. To estimate gravity effects we used two types of cultured stem cells: human mesenchymal stromal cells (hMSCs) from bone marrow and mice embryonic stem (mESC) line R1. Gravity changes were simulated by long-term (4-7 days) slow clinorotation and leaded to decreased hMSC proliferation, changes of cell morphology and modified F-actin cytoskeleton. We did not find the shifts in cell phenotype except for decreased expression of HLA 1 and CD105 but excretion of IL-6 into medium increased significantly. Remodeling of cytoskeleton started after first 4 h and was similar to preapoptotic changes. This data suggested the modification in cell adhesion and possible commitment of hMSC. It was observed that expression of alkaline phosphatase by MSC in osteogenic medium was more intensive in control. On the contrary, clinorotation did not change formation of mESC colonies and increased proliferation activity in LIF+-medium. However, the number of embryonic bodies after clinorotation was less than in static control. It is suggested that ESCs kept the viability and proliferative potential but decreased the differentiation ability after changes in gravity stimulation.

  5. In vitro propagation of Morus alba L. in semisolid culture medium.

    Directory of Open Access Journals (Sweden)

    Enrique Salas Barbosa

    2005-04-01

    Full Text Available Apical buds as explants were used with the objective to propagate in vitro mulberry plants in semisolid MS culture medium suplemented with 6-BAP and KIN in their establishment and, with different combinations of 6-BAP with ANA in the multiplication phase. In vitro plants were evaluated during the acclimatization phase. It is necessary to supplement the basal MS culture media with 0.5 mg.l-1 of 6-BAP to induce the sprouting and, 0.5 mg.l-1 of 6-BAP and 0.5 mg.l-1 of ANA to multiply the mulberry by nodal segments. In the acclimatization phase a 95% of survival, 30.2 cm of height, 9.8 leaves and 2.02 g.plant-1 of dry mass was observed. In vitro propagation of mulberry was achieved as an alternative for plants production. Key words: acclimatization, apical buds, establishment, explant, shooting

  6. Detection of microorganisms in culture medium through the neutron radiographic technique

    International Nuclear Information System (INIS)

    Wacha, Reinaldo

    1999-05-01

    The study aims to obtain a more effective and faster method for the detection of bacteria in several culture media, such as potable water and blood. After the process growth in the culture medium, separation and suspension in buffer solution based in boron, the bacteria are deposited in track detectors that are submitted to thermal neutron beams (neutron flux: 2,2 x 10 5 n.cm -2 .s -1 ), resulting from the channel J-9 of the Argonauta research reactor, from the Nuclear Engineering Institute, IEN/CNEN. The latent tracks arisen from the alpha particles proceeding from the reaction B (η, α) Li and, after having been revealed, are analyzed by an optical microscope that allows to detect the existence of the bacteria. Afterwards, they were analyzed in a nanoscope which helps the identification of the tracks of the alpha particles. (author)

  7. Optimization of culture medium for heavy-ion irradiation bread yeast design

    International Nuclear Information System (INIS)

    Ma Liang; Wang Jufang; Lu Dong; Li Wenjian; Xiao Guoqing

    2013-01-01

    A mutant bread yeast strain with high protein content of 55% was gained by use of 12 C 6+ ions. The MINITAB 16.0 software, Plackett-Burman experimental design and response surface methodology were applied to optimize the culture medium for the irradiated yeast. The most important three factors which influenced the culture results were identified as glucose, magnesium sulphate and yeast extract. The path of the steepest ascent was undertaken to approach the optimal region of the three significant factors. Box-Behnken design and response surface methodology were used for the regression analysis. Finally, the optimal fermentation conditions were identified as glucose 11.03 g/L, yeast extract 6.53 g/L and magnesium sulphate 5.59 g/L by the regression analysis. It was found that the biomass of the bread yeasts reached 4.84 g/L and increased by 15% compared to original conditions. (authors)

  8. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    International Nuclear Information System (INIS)

    Fan, Ping; He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan

    2011-01-01

    Research highlights: → The proliferation of dramatic increased by co-cultured with Sertoli cells. → VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. → The MHC expression of ECs induced by INF-γ and IL-6, IL-8 and sICAM induced by TNF-α decreased respectively after co-cultured with Sertoli cells. → ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10 3 , 1 x 10 4 or 1 x 10 5 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10 4 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P 4 cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single

  9. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli

  10. Regulation of the efflux of putrescine and cadaverine from rapidly growing cultured RAW 264 cells by extracellular putrescine.

    OpenAIRE

    Tjandrawinata, R R; Byus, C V

    1995-01-01

    Cultures of the macrophage-like RAW 264 cells were adapted to divide normally in a synthetic serum-supplemented culture medium lacking any polyamines and diamine oxidase activity. These rapidly dividing cells actively effluxed large amounts of putrescine and cadaverine, compared with the intracellular levels, into the culture medium. The efflux of putrescine was stimulated by the amino acid ornithine, whereas efflux of cadaverine was inhibited. Relatively low levels of spermidine and N1-acety...

  11. In Vitro Selection of Peanut Somatic Embryos on Medium Containing Culture Filtrate of Sclerotium rolfsii and Plantlet Regeneration

    Directory of Open Access Journals (Sweden)

    YUSNITA

    2005-06-01

    Full Text Available Attempts to identify somaclonal variants of peanut with resistance to Sclerotium stem rot disease due to infection of S. rolfsii were conducted. The objectives of this study were to develop in vitro selection method using culture filtrates of S. rolfsii, identify culture filtrate-insensitive somatic embryo (SE of peanut after in vitro selection and regenerate peanut R0 lines originated from culture filtrate-insensitive SE. To achieve these objectives, peanut embryogenic tissues were cultured on selective medium containing various concentrations of S. rolfsii culture filtrates and sublethal concentration of the filtrates. Medium containing sublethal level of S. rolfsii culture filtrates was used to identify culture filtrate-insensitive SE of peanut. Subsequently, the selected SEs were germinated, plantlets were regenerated and preliminary tested against S. rolfsii. Results of the experiments showed that addition of S. rolfsii culture filtrates into medium for inducing peanut somatic embryos drastically reduced their growth and proliferation. S. rolfsii culture filtrates at 10% concentration has significantly reduced the number of proliferated SE per explant. However, sublethal level was achieved at 30% of culture filtrates concentration. Responses of five peanut cultivars against 30% of culture filtrates were similar, indicating they were similar in their susceptibility against S. rolfsii. A number of culture filtrate-insensitive SE were identified after culturing 1500 clumps of embryogenic tissue of peanut cv. Kelinci for three consecutive passages on medium containing 30% of culture filtrates. Germination of selected SE and regeneration of plantlet from culture filtrate-insensitive SE resulted in 50 peanut R0 lines. These lines have been grown in the plastic house and produced normal seeds for further evaluation. Results of S. rolfsii inoculation indicated the existence of chimera for insensitivity against S. rolfsii.

  12. Accumulation of {sup 137}Cs in trefoil (leaf and stem), ``Mitsuba``, Cryptotaenia japonica Hassk, immersed in hydroponic culture medium

    Energy Technology Data Exchange (ETDEWEB)

    Motegi, Misako; Miyake, Sadaaki; Ohsawa, Takashi; Nakazawa, Kiyoaki [Saitama Institute of Public Health, Urawa (Japan); Izumo, Yoshiro

    1998-11-01

    Accumulation of {sup 137}Cs in trefoil (leaf and stem), ``Mitsuba``, Cryptotaenia japonica Hassk, with or without root was investigated to prepare higher radioactive plant in hydroponic culture medium (140-150 Bq/ml). It was found that {sup 137}Cs concentration in plant tissue was increased with time, as high as 1.6 times of that in the culture medium after 4 days. On the other hand, {sup 137}Cs concentration was affected by carrier element (Cs>6 ppm) and coexistent elements in the medium. Radioactivity of the plant after 4 days was shown to be sufficient for successive experiments. (author)

  13. Accumulation of 137Cs in trefoil (leaf and stem), ''Mitsuba'', Cryptotaenia japonica Hassk, immersed in hydroponic culture medium

    International Nuclear Information System (INIS)

    Motegi, Misako; Miyake, Sadaaki; Ohsawa, Takashi; Nakazawa, Kiyoaki; Izumo, Yoshiro

    1998-01-01

    Accumulation of 137 Cs in trefoil (leaf and stem), ''Mitsuba'', Cryptotaenia japonica Hassk, with or without root was investigated to prepare higher radioactive plant in hydroponic culture medium (140-150 Bq/ml). It was found that 137 Cs concentration in plant tissue was increased with time, as high as 1.6 times of that in the culture medium after 4 days. On the other hand, 137 Cs concentration was affected by carrier element (Cs>6 ppm) and coexistent elements in the medium. Radioactivity of the plant after 4 days was shown to be sufficient for successive experiments. (author)

  14. Treatment of mycoplasma contamination in cell cultures with Plasmocin.

    Science.gov (United States)

    Uphoff, Cord C; Denkmann, Sabine-A; Drexler, Hans G

    2012-01-01

    A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

  15. Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

    Directory of Open Access Journals (Sweden)

    Cord C. Uphoff

    2012-01-01

    Full Text Available A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78% mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

  16. Dynamic analysis of reactive oxygen nitrogen species in plasma-activated culture medium by UV absorption spectroscopy

    Science.gov (United States)

    Brubaker, Timothy R.; Ishikawa, Kenji; Takeda, Keigo; Oh, Jun-Seok; Kondo, Hiroki; Hashizume, Hiroshi; Tanaka, Hiromasa; Knecht, Sean D.; Bilén, Sven G.; Hori, Masaru

    2017-12-01

    The liquid-phase chemical kinetics of a cell culture basal medium during treatment by an argon-fed, non-equilibrium atmospheric-pressure plasma source were investigated using real-time ultraviolet absorption spectroscopy and colorimetric assays. Depth- and time-resolved NO2- and NO3- concentrations were strongly inhomogeneous and primarily driven by convection during and after plasma-liquid interactions. H2O2 concentrations determined from deconvolved optical depth spectra were found to compensate for the optical depth spectra of excluded reactive species and changes in dissolved gas content. Plasma-activated media remained weakly basic due to NaHCO3 buffering, preventing the H+-catalyzed decomposition of NO2- seen in acidic plasma-activated water. An initial increase in pH may indicate CO2 sparging. Furthermore, the pH-dependency of UV optical depth spectra illustrated the need for pH compensation in the fitting of optical depth data.

  17. Statistical Modelling and Optimization of Fermentation Medium for Lincomycin Production by Streptomyces lincolnensis Immobilized Cells

    Directory of Open Access Journals (Sweden)

    Nayera A.M. Abdelwahed

    2017-05-01

    Full Text Available ABSTRACT Response surface methodology was used to optimize lincomycin production by Streptomyces lincolnensis NRRL ISP-5355 in submerged fermentation. Screening of fermentation medium components to find their relative effect on lincomycin production was done using Plackett-Burman design. Malt extract, dextrin, soluble starch and (NH42SO4 were the most significant nutrient influenced on lincomycin production. Central composite design was applied to determine optimal concentrations of these factors and the effect of their mutual interactions. The interaction between soluble starch and (NH42SO4 was found to enhance the production, whereas malt extract and dextrin exhibited an influence independent from the other two factors. Using this statistical optimization method, maximum lincomycin concentration of 1345 μg/ml was obtained which represented a 40.5 % increase in titer than that acquired from the non-optimized medium. This statistically optimized medium was employed for lincomycin production through immobilization of Streptomyces lincolnensis by adsorption on synthetic cotton fibers. Immobilization technique improved the concentration to 1350 μg/ml higher than that produced from free cells cultures and could be maintained for longer than 17 days in a repeated batch system.

  18. Neuron-specific enolase is a useful maker of neuroendocrine origin in pheochromocytoma cell culture

    International Nuclear Information System (INIS)

    Abelin, N.; Dahia, P.L.M.; Martin, R.; Kato, S.; Toledo, S.P.A.

    1994-01-01

    Neuron-specific enolase (NSE) has been used as a marker for neuroendocrine tumors either in immunocytochemical studies or in serum measurements. In this paper NSE levels were determined in cultured pheochromocytoma cells to test whether it is also a useful marker in cell culture of tumors derived from neuroendocrine system. Cultured pheochromocytoma cells came from a primary explant and were grown in RPMI supplemented with 20% fetal calf serum, 100 μg/mL ampicillin and 100 μ/mL streptomycin. NSE was measured in culture medium and cell homogenates. Samples from different pheochromocytoma cultures were analyzed and compared to normal cultured fibroblast cells derived from human skin. NSE was measured by a commercially available radioimmunoassay kit. NSE levels were higher in cell homogenates as compared to those in culture medium, reaching levels as high as 6-fold in the former in TE cell line (26.46 ng/mL and 4.39 ng/mL, respectively). Serial measurements in culture medium from TE cell line evidenced decreasing values in subsequential subcultures (from 9.24 ng/mL during primary explant to 1.7 ng/mL in the tenth subculture). In cultured normal fibroblasts, NSE levels in cultured media were definitely lower than those obtained from pheochromocytoma cultures. These preliminary data suggest that NSE may be a useful marker of neuroendocrine derived tumors, such as pheochromocytoma, in culture. Thus, the simplicity and availability of NSE radioimmunoassay provides an alternative to catecholamine measurement to better characterize pheochromocytoma cell lines in culture, with the advantage of faster result at lower costs. (author). 18 refs, 2 tabs

  19. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  20. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Science.gov (United States)

    Mass, Tali; Drake, Jeana L; Haramaty, Liti; Rosenthal, Yair; Schofield, Oscar M E; Sherrell, Robert M; Falkowski, Paul G

    2012-01-01

    The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  1. Aragonite precipitation by "proto-polyps" in coral cell cultures.

    Directory of Open Access Journals (Sweden)

    Tali Mass

    Full Text Available The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag~4, the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

  2. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  3. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF

    NARCIS (Netherlands)

    Vergouw, C.G.; Kostelijk, E.H.; Doejaaren, E.; Hompes, P.G.A.; Lambalk, C.B.; Schats, R.

    2012-01-01

    STUDY QUESTION Does the type of medium used to culture fresh and frozenthawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? SUMMARY ANSWER A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean

  4. Effects of culture medium compositions on antidiabetic activity and anticancer activity of marine endophitic bacteria isolated from sponge

    Science.gov (United States)

    Maryani, Faiza; Mulyani, Hani; Artanti, Nina; Udin, Linar Zalinar; Dewi, Rizna Triana; Hanafi, Muhammad; Murniasih, Tutik

    2017-01-01

    High diversity of Indonesia marine spesies and their ability in producing secondary metabolite that can be used as a drug candidate cause this fascinating topic need to explore. Most of marine organisms explored to discover drug is macroorganism whereas microorganism (such as Indonesia marine bacteria) is very limited. Therefore, in this report, antidiabetic and anticancer activity of Indonesia marine bacteria isolated from Sponges's extract have been studied. Bacteria strain 8.9 which are collection of Research Center for Oseanography, Indonesian Institute of Sciences were from Barrang Lompo Island, Makasar, Indonesia. Bacteria were cultured in different culture medium compositions (such as: different pH, source of glucose and water) for 48 hours on a shaker, then they were extracted with ethyl asetate. Extracts of bacteria were tested by DPPH method (antioxidant activity), alpha glucosidase inhibitory activity method (antidiabetic activity), and Alamar Blue assay (anticancer activity) at 200 ppm. According to result, extract of bacteria in pH 8.0 exhibited the greatest antioxidant (19.27% inhibition), antidiabetic (63.95% inhibition) and anticancer activity of T47D cell line (44.62% cell viability) compared to other extracts. However, effect of addition of sugar sources (such as: glucose, sucrose, and soluble starch) and effect of addition of water/sea water exhibited less influence on their bioactivities. In conclusion, Indonesia marine bacteria isolated from sponge have potential a source of bioactive compound in drug discovery field.

  5. Isolation and culture of pulmonary endothelial cells.

    OpenAIRE

    Ryan, U S

    1984-01-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vas...

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

    Directory of Open Access Journals (Sweden)

    Retno Mastuti

    2003-06-01

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

  7. Cell Culture in Microgravity: Opening the Door to Space Cell Biology

    Science.gov (United States)

    Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Adaptational response of human cell populations to microgravity is investigated using simulation, short-term Shuttle experiments, and long-term microgravity. Simulation consists of a clinostatically-rotated cell culture system. The system is a horizontally-rotated cylinder completely filled with culture medium. Low speed rotation results in continuous-fall of the cells through the fluid medium. In this setting, cells: 1) aggregate, 2) propagate in three dimensions, 3) synthesize matrix, 4) differentiate, and 5) form sinusoids that facilitate mass transfer. Space cell culture is conducted in flight bioreactors and in static incubators. Cells grown in microgravity are: bovine cartilage, promyelocytic leukemia, kidney proximal tubule cells, adrenal medulla, breast and colon cancer, and endothelium. Cells were cultured in space to test specific hypotheses. Cartilage cells were used to determine structural differences in cartilage grown in space compared to ground-based bioreactors. Results from a 130-day experiment on Mir revealed that cartilage grown in space was substantially more compressible due to insufficient glycosaminoglycan in the matrix. Interestingly, earth-grown cartilage conformed better to the dimensions of the scaffolding material, while the Mir specimens were spherical. The other cell populations are currently being analyzed for cell surface properties, gene expression, and differentiation. Results suggest that some cells spontaneously differentiate in microgravity. Additionally, vast changes in gene expression may occur in response to microgravity. In conclusion, the transition to microgravity may constitute a physical perturbation in cells resulting in unique gene expressions, the consequences of which may be useful in tissue engineering, disease modeling, and space cell biology.

  8. Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements.

    Science.gov (United States)

    Wang, Chien-Yuan; Chen, Ling-Lan; Kuo, Pei-Yin; Chang, Jia-Ling; Wang, Yng-Jiin; Hung, Shih-Chieh

    2010-04-01

    Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.

  9. Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

    Directory of Open Access Journals (Sweden)

    Angelique eLewies

    2014-07-01

    Full Text Available The comet assay is a simple and cost effective technique, commonly used to analyse and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions.

  10. The Effect of Culture Medium on Metabolic and Antibacterial Activities of Probiotic Bacteria

    Directory of Open Access Journals (Sweden)

    f Mirdavoudi

    2012-05-01

    Full Text Available

    Background and Objectives: Probiotic bacteria is added directly to food components and it has beneficial effect on function and the health of organisms. The bifidogenic factors enter the colon where they contribute to an increase lactic acid bacteria population including Lactobacilli and Bifidobacteria and they inhibit enteric pathogenic bacterial growth. The aim of this study is to investigate the effect of culture medium on metabolic and antibacterial of probiotic bacteria.

     

    Methods: In this study, the probiotics bacterial and intestine pathogenic are to be used. Lactobacilli and Bifidobacterium were identified by plating samples on MRS medium, Gram Staining and standard biochemical methods. The effect of antagonistic probiotics was investigated in the presence of growth factor in the method well diffusion Ager on the Shigella flexneri (PTCC 1234, Escherichia coli (PTCC 1552, Salmonella typhi ( PTCC 1609 and the culture medium pH was measured.

     

    Results: The probiotics bacterial growth in MRS and lactose1%, sorbitol, raffinose, riboflavin were shown the effect antibacterial. The results of the study show the most antagonistic activity in commercial strain Lactobacillus acidophilus on Shigella flexneri and lower activity was in Lactobacillus casei (PTCC 1608, and Salmonella typhimurium (PTCC 1609, and also in Bbifidobacterium bifidum, it showed the most decrease pH value.

     

    Conclusion: According to the result of the study, adding growth factors to MRS medium base and lactose 1%, probiotic growth was increased and which also increased antagonistic activity.

     

  11. Day-3 Medium Changes can Affect Developmental Potential of Porcine Somatic Cell Nuclear Transfer and Parthenogenesis Embryos In Vitro

    Directory of Open Access Journals (Sweden)

    Dibyendu Biswas and Sang Hwan Hyun*

    2011-01-01

    Full Text Available The aim of the present study was to compare the developmental competence of porcine parthenotes and somatic cell nuclear transfer (SCNT embryos after day-3 medium change with fresh embryo culture medium to that of embryos that did not have a medium change (monoculture system. The parthenogenetic and SCNT blastocyst formation rates were significantly (P<0.05 higher in the no-medium-change group (43.3±2.3, 18.5±1.1%, respectively compared with the day-3 medium-change group (35.9±2.4, 7.9±0.9%, respectively. Total cell number in parthenotes and SCNT blastocysts was also significantly (P<0.05 higher in the no-media-change group (92.0±4.2, 66.9±7.7, respectively compared with the media-change group (81.5±3.1, 46.6±4.9, respectively. No significant difference in cleavage rate was found in either group for parthenotes or SCNT embryos. This result suggests that day-3 medium changes have negative effects on porcine parthenotes and SCNT embryos in vitro.

  12. Optimization of culture medium and modeling of curdlan production from Paenibacillus polymyxa by RSM and ANN.

    Science.gov (United States)

    Rafigh, Sayyid Mahdi; Yazdi, Ali Vaziri; Vossoughi, Manouchehr; Safekordi, Ali Akbar; Ardjmand, Mehdi

    2014-09-01

    Paenibacillus polymyxa ATCC 21830 was used for the production of curdlan gum for first time. A Box-Behnken experimental design was applied to optimize six variables of batch fermentation culture each at three levels. Statistical analyses were employed to investigate the direct and interactive effects of variables on curdlan production. Optimum cultural conditions were temperature (50°C), pH (7), fermentation time (96 h), glucose (100 g/L), yeast extract (3 g/L) and agitation speed (150 rpm). The yield of curdlan production was 6.89 g/L at optimum condition medium. Response surface methodology (RSM) and artificial neural network (ANN) were used to model cultural conditions of curdlan production. The maximum yield of curdlan production were predicted to be 6.68 and 6.85 g/L by RSM and ANN at optimum condition. The prediction capabilities of RSM and ANN were then statistically compared. The results showed that the ANN model is much more accurate in prediction as compared to the RSM. The infrared (IR) and NMR spectra, the thermogram of DSC and pattern of X-ray diffraction for the curdlan of the present study were almost identical to those of the commercial curdlan sample. The average molecular weight of the purified curdlan was determined to be 170 kDa by gel permeation chromatography. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    International Nuclear Information System (INIS)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3 H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3 H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture

  14. Metabolome analysis of Saccharomyces cerevisiae and optimization of culture medium for S-adenosyl-L-methionine production.

    Science.gov (United States)

    Hayakawa, Kenshi; Matsuda, Fumio; Shimizu, Hiroshi

    2016-12-01

    S-Adenosyl-L-methionine (SAM) is a fine chemical used as a nutritional supplement and a prescription drug. It is industrially produced using Saccharomyces cerevisiae owing to its high SAM content. To investigate the optimization of culture medium components for higher SAM production, metabolome analysis was conducted to compare the intracellular metabolite concentrations between Kyokai no. 6 (high SAM-producing) and laboratory yeast S288C (control) under different SAM production conditions. Metabolome analysis and the result of principal component analysis showed that the rate-limiting step for SAM production was ATP supply and the levels of degradation products of adenosine nucleotides were higher in Kyokai 6 strain than in the S288C strain under the L-methionine supplemented condition. Analysis of ATP accumulation showed that the levels of intracellular ATP in the Kyokai 6 strain were also higher compared to those in the S288C strain. Furthermore, as expected from metabolome analysis, the SAM content of Kyokai 6 strain cultivated in the medium without yeast extract increased by 2.5-fold compared to that in the additional condition, by increasing intracellular ATP level with inhibited cell growth. These results suggest that high SAM production is attributed to the enhanced ATP supply with L-methionine condition and high efficiency of intracellular ATP consumption.

  15. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  16. Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM.

    Science.gov (United States)

    Peng, Yanjie; He, Yanhui; Wu, Zhansheng; Lu, Jianjiang; Li, Chun

    2014-01-01

    The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K₂HPO₄, 0.19 g/L MnSO₄ and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K₂HPO₄, 0.17 g/L MnSO₄ and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

  17. The kinetics of Escherichia coli B growth and bacteriophage T4 multiplication in SM-1 novel minimal culture medium.

    Science.gov (United States)

    Sochocka, Marta; Tomczyk, Tomasz; Sobczyński, Maciej; Szermer-Olearnik, Bożena; Boratyński, Janusz

    2015-01-01

    The aim of this study was to develop a minimal medium for the cultivation of Escherichia coli B, which could be especially suitable for the industrial propagation of bacteriophage T4. The new defined, minimal SM-1 culture medium, contains free amino acids as the only nitrogen source and enables the bacteria generation time to be prolonged and satisfactory phage titers to be achieved. The presence of organic ingredients, such as meat extracts, yeast hydrolysates, enzymatic protein hydrolysates, in a culture medium may cause problems in the case of bacteria or phage cultures for therapeutic purposes. In the present study, we introduce a new medium, together with some procedures and applications for its usage. We also present new kinetics of E. coli B growth. Some traits such as the lack of high molecular proteins, a bacterial growth comparable to that in a rich medium, and the cost effectiveness of the medium, makes it highly competitive with currently used microbiological media. The surprisingly high titers of bacteriophage T4 obtained in our experiments suggest that SM-1 medium has the potential to find a broad application in medicine, especially in infectious disease therapy, pharmacy and biotechnology.

  18. [Solubility of metal components into tissue culture medium from dental amalgams (author's transl)].

    Science.gov (United States)

    Kawahara, H; Yamada, T; Nakamura, M; Tomoda, T; Kobayashi, H; Saijo, A; Kawata, Y; Hikari, S

    1981-10-01

    Solubility of metal components into tissue culture medium, YLH, from various dental amalgams including high copper, conventional and copper amalgam was measured with atomic absorption spectrometry. The results obtained were as follows.: 1. Mercury solubility was found much in all the dental amalgams after one day extraction. This was followed by gradual increase in solubility in the high copper amalgams until after seven days. On the otherhand, initial high solubility was maintained in conventional and copper amalgams through the whole experimental period. 2. Higher solubility of silver and copper was recorded in the high copper amalgams. 3. Zinc was only found in Dispersalloy and copper amalgam. 4. Tin was unable to be measured in all the amalgams examined. It was considered that the present results could shed a light on the mechanism for cytotoxicity yielding of the dental amalgams.

  19. Cultured meat from stem cells: challenges and prospects.

    Science.gov (United States)

    Post, Mark J

    2012-11-01

    As one of the alternatives for livestock meat production, in vitro culturing of meat is currently studied. The generation of bio-artificial muscles from satellite cells has been ongoing for about 15 years, but has never been used for generation of meat, while it already is a great source of animal protein. In order to serve as a credible alternative to livestock meat, lab or factory grown meat should be efficiently produced and should mimic meat in all of its physical sensations, such as visual appearance, smell, texture and of course, taste. This is a formidable challenge even though all the technologies to create skeletal muscle and fat tissue have been developed and tested. The efficient culture of meat will primarily depend on culture conditions such as the source of medium and its composition. Protein synthesis by cultured skeletal muscle cells should further be maximized by finding the optimal combination of biochemical and physical conditions for the cells. Many of these variables are known, but their interactions are numerous and need to be mapped. This involves a systematic, if not systems, approach. Given the urgency of the problems that the meat industry is facing, this endeavor is worth undertaking. As an additional benefit, culturing meat may provide opportunities for production of novel and healthier products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.

    Science.gov (United States)

    Yamasaki, Sachiko; Nabeshima, Kou; Sotomaru, Yusuke; Taguchi, Yuki; Mukasa, Hanae; Furue, Miho K; Sato, J Denry; Okamoto, Tetsuji

    2013-01-01

    Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

  1. Oxidative Stress Induces Senescence in Cultured RPE Cells.

    Science.gov (United States)

    Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

    2016-01-01

    The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

  2. New culture medium for the presumptive identificaion of Candida albicans and Cryptococcus neoformans.

    Science.gov (United States)

    Fleming, W H; Hopkins, J M; Land, G A

    1977-02-01

    A new medium composed of Tween 80, oxgall, caffeic acid, and Davis agar (TOC) that provides for the rapid presumptive identification of Candida albicans and Cryptococcus neoformans is described herein. C. albicans is differentiated from other yeasts by the sequential production of germ tubes and chlamydospores. In a comparison with cormeal agar control plates, there was an increase of chlamydospore-forming strains of C. albicans (97.1% versus 87.2%) and a decrease in the time required for chlamydospore formation (24 h versus 48 h). C. neoformans produced a brown pigment of TOC, which is specific for its identification, thus differentiating it from the other yeasts. A comparison of 24-h pigment production by C. neoformans on TOC with that of birdseed agar showed a dark, coffee brown color in the former cultures and a light brown color in the latter. The change in pigmentation of C. neoformans, as well as morphological changes in C. albicans, can be induced within 3 to 12 h and in not more than 24 h on the TOC medium.

  3. Optimization of Culture Medium for Lactobacillus bulgaricus using Box-Behnken Design

    Directory of Open Access Journals (Sweden)

    Zhang Bowen

    2017-06-01

    Full Text Available Lactobacillus bulgaricus is a common yogurt starter in dairy production. But the viable counts of the bacteria in the productions are relatively low during free-drying and storage which is not good for its commercial production. In order to obtain a medium with high activity and high density for bacterial cultured, the experiments and regression analysis were conducted by Box-Behnken design in this study, and a model was established to predict the influence of glucose (9-11 g·L−1, casein hydrolysate (15-17 g·L−1 and glutamate (6.5-7.5 mg·L−1 on viable counts of L. bulgaricus and. The results showed that the glucose, 9.5 g·L−1; casein hydrolysate, 15.5 g·L−1; glutamate, 7.0mg·L−1, the number of viable bacteria of L. bulgaricus could reach (2.95±0.07 ×109, which was very similar to the predicted value of the model of 3.00×109 cfu·mL−1, indicating that the optimized conditions and models used were feasible and effective. The optimized medium components can improve the viable counts of bacteria which are useful from its application in industrial production.

  4. Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

    Directory of Open Access Journals (Sweden)

    Alan Yung-Chih Hu

    Full Text Available BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK cells grown in a serum-free (SF medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

  5. Radiation-induced bystander effects in cultured human stem cells.

    Directory of Open Access Journals (Sweden)

    Mykyta V Sokolov

    2010-12-01

    Full Text Available The radiation-induced "bystander effect" (RIBE was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR. RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.Human bone-marrow mesenchymal stem cells (hMSC and embryonic stem cells (hESC were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05. A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05.These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies.

  6. [Biological characteristics of mesenchymal stem cell and hematopoietic stem cell in the co-culture system].

    Science.gov (United States)

    Wei, Wei; Xu, Chao; Ye, Zhi-Yong; Huang, Xiao-Jun; Yuan, Jia-En; Ma, Tian-Bao; Lin, Han-Biao; Chen, Xiu-Qiong

    2016-10-25

    The aim of the present study was to obtain the qualified hematopoietic stem/progenitor cells (HSC/HPC) and human umbilical cord-mesenchymal stem cells (MSC) in vitro in the co-culture system. Cord blood mononuclear cells were separated from umbilical cord blood by Ficoll lymphocyte separation medium, and then CD34 + HSC was collected by MACS immunomagnetic beads. The selected CD34 + HSC/HPC and MSC were transferred into culture flask. IMDM culture medium with 15% AB-type cord plasma supplemented with interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt-3L) factors were used as the co-culture system for the amplification of HSC/HPC and MSC. The cellular growth status and proliferation on day 6 and 10 after co-culture were observed by using inverted microscope. The percentage of positive expression of CD34 in HSC/HPC, as well as the percentages of positive expressions of CD105, CD90, CD73, CD45, CD34 and HLA-DR in the 4 th generation MSC, was tested by flow cytometry. Semisolid colony culture was used to test the HSC/HPC colony forming ability. The osteogenic, chondrogenesis and adipogenic ability of the 4 th generation MSC were assessed. The karyotype analysis of MSC was conducted by colchicines. The results demonstrated that the HSC/HPC of co-culture group showed higher ability of amplification, CFU-GM and higher CD34 + percentage compared with the control group. The co-cultured MSC maintained the ability to differentiate into bone cells, fat cells and chondrocytes. And the karyotype stability of MSC remained normal. These results reveal that the appropriate co-culture system for MSC and HSC is developed, and via this co-culture system we could gain both two kinds of these cells. The MSCs under the co-culture system maintain the biological characteristics. The CFU-GM ability, cell counting and the flow cytometry results of HSC/HPC under the co-culture system are conform to the criterion, showing that

  7. Characteristics of plasma in culture medium generated by positive pulse voltage and effects of organic compounds on its characteristics

    Science.gov (United States)

    Sato, Y.; Sato, T.; Yoshino, D.

    2016-12-01

    We describe a positive pulse voltage method for generating plasma in culture medium with a composition similar to biological fluids. We also describe the plasma’s characteristics, liquid quality, and the effect of organic compounds in the culture medium on the plasma characteristics through comparisons to a solution containing inorganic salts at the same concentrations as in the culture medium. Light emission with Na and OH spectra was observed within a vapor bubble produced by Joule heating at the tip of the electrode. A downward thermal flow and shock wave were caused by the behavior of the vapor bubble. The culture medium pH gradually increased from 7.9 to 8.3 over the discharge time of 300 s. H2O2 was generated 1.1 mg l-1 in the culture medium after discharge for 300 s, and this value was 0.5 mg l-1 lower than the inorganic salts solution which does not contain organic compounds. This study provides important data that will help facilitate more widespread application of plasma medicine.

  8. Scedo-Select III: a new semi-selective culture medium for detection of the Scedosporium apiospermum species complex.

    Science.gov (United States)

    Pham, Trâm; Giraud, Sandrine; Schuliar, Gaëlle; Rougeron, Amandine; Bouchara, Jean-Philippe

    2015-06-01

    The Scedosporium apiospermum complex is responsible for a large variety of infections in human. Members of this complex have become emerging fungal pathogens with an increasing occurrence in patients with underlying conditions such as immunosuppression or cystic fibrosis. A better knowledge of these fungi and of the sources of contamination of the patients is required and more accurate detection methods from the environment are needed. In this context, a highly selective culture medium was developed in the present study. Thus, various aliphatic, cyclic, or aromatic compounds were tested as the sole carbon source, in combination with some inorganic nitrogen sources and fungicides. The best results were obtained with 4-hydroxy-benzoate combined with ammonium sulfate and the fungicides dichloran and benomyl. This new culture medium called Scedo-Select III was shown to support growth of all species of the S. apiospermum complex. Subsequently, this new culture medium was evaluated successfully on water and soil samples, exhibiting higher sensitivity and selectivity than the previously described SceSel+ culture medium. Therefore, this easy-to-prepare and synthetic semi-selective culture medium may be useful to clarify the ecology of these fungi and to identify their reservoirs in patients' environment. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Design and Performance of an Automated Bioreactor for Cell Culture Experiments in a Microgravity Environment

    Directory of Open Access Journals (Sweden)

    Youn-Kyu Kim

    2015-03-01

    Full Text Available In this paper, we describe the development of a bioreactor for a cell-culture experiment on the International Space Station (ISS. The bioreactor is an experimental device for culturing mouse muscle cells in a microgravity environment. The purpose of the experiment was to assess the impact of microgravity on the muscles to address the possibility of longterm human residence in space. After investigation of previously developed bioreactors, and analysis of the requirements for microgravity cell culture experiments, a bioreactor design is herein proposed that is able to automatically culture 32 samples simultaneously. This reactor design is capable of automatic control of temperature, humidity, and culture-medium injection rate; and satisfies the interface requirements of the ISS. Since bioreactors are vulnerable to cell contamination, the medium-circulation modules were designed to be a completely replaceable, in order to reuse the bioreactor after each experiment. The bioreactor control system is designed to circulate culture media to 32 culture chambers at a maximum speed of 1 ml/min, to maintain the temperature of the reactor at 36±1°C, and to keep the relative humidity of the reactor above 70%. Because bubbles in the culture media negatively affect cell culture, a de-bubbler unit was provided to eliminate such bubbles. A working model of the reactor was built according to the new design, to verify its performance, and was used to perform a cell culture experiment that confirmed the feasibility of this device.

  10. Design and Performance of an Automated Bioreactor for Cell Culture Experiments in a Microgravity Environment

    Science.gov (United States)

    Kim, Youn-Kyu; Park, Seul-Hyun; Lee, Joo-Hee; Choi, Gi-Hyuk

    2015-03-01

    In this paper, we describe the development of a bioreactor for a cell-culture experiment on the International Space Station (ISS). The bioreactor is an experimental device for culturing mouse muscle cells in a microgravity environment. The purpose of the experiment was to assess the impact of microgravity on the muscles to address the possibility of longterm human residence in space. After investigation of previously developed bioreactors, and analysis of the requirements for microgravity cell culture experiments, a bioreactor design is herein proposed that is able to automatically culture 32 samples simultaneously. This reactor design is capable of automatic control of temperature, humidity, and culture-medium injection rate; and satisfies the interface requirements of the ISS. Since bioreactors are vulnerable to cell contamination, the medium-circulation modules were designed to be a completely replaceable, in order to reuse the bioreactor after each experiment. The bioreactor control system is designed to circulate culture media to 32 culture chambers at a maximum speed of 1 ml/min, to maintain the temperature of the reactor at 36°C, and to keep the relative humidity of the reactor above 70%. Because bubbles in the culture media negatively affect cell culture, a de-bubbler unit was provided to eliminate such bubbles. A working model of the reactor was built according to the new design, to verify its performance, and was used to perform a cell culture experiment that confirmed the feasibility of this device.

  11. Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.

    Directory of Open Access Journals (Sweden)

    Makiko Nakahara

    Full Text Available Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs, even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM obtained from human bone marrow-derived mesenchymal stem cells (MSCs (MSC-CM for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+/K(+-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1 for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary

  12. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  13. Metabolism of 4-nitrobiphenyl (NBP) by cultured rat urothelial cells

    International Nuclear Information System (INIS)

    Swaminathan, S.; Lang, D.B.; Reznikoff, C.A.

    1986-01-01

    The potential of rat urothelial cells to metabolize NBP was evaluated by incubating 4.3 x 10 7 viable cells with 20 μM [ 3 H]NBP in a serum free medium for 48 hours. The culture medium was examined for metabolites of NBP by extraction with ethyl acetate and subsequent chromatographic analysis. High pressure liquid chromatography of the solvent extract using a Whatman ODS-3, C-18 column in 70% methanol-water at a flow rate of 1 ml/min revealed two major peaks at retention times of approximately 8 and 13 min. Thin layer chromatography showed two regions of radioactivity at Rf values of 0.35 and 0.83, the latter corresponding with NBP. Based on the chromatographic data the metabolite with the retention time of 8.0 min in HPLC and an Rf of 0.35 in TLC has been tentatively identified as 4-acetylaminobiphenyl. Analysis of binding to proteins and nucleic acids following exposure to [ 3 H]NBP revealed a significant amount (0.03% of initially applied radioactivity) in the protein fractions. Control samples of NBP incubated in medium, without the urothelial cells revealed only the parent compound. These data suggest that rat bladder cells possess the metabolic capability to reduce NBP and to generate reactive metabolites that bind to cellular macromolecules

  14. Effects of cultural medium and conditions on the proliferation and hypoglycemic activity of Saccharomyces pastorianus no. 54.

    Science.gov (United States)

    Wu, Chien-Hui; Lin, Hong-Ting; Wu, Guan-James; Wang, Sheng-Hong; Tsai, Guo-Jane

    2011-08-01

    A yeast strain of Saccharomyces pastorianus no. 54 with hypoglycemic activity was isolated from soils of a winery. The aims of this study were first to investigate the effects of the cultivation conditions on proliferation and hypoglycemic activity of this yeast using the assay model of the differentiated 3T3-L1 adipocytes, and then, to confirm in vivo the hypoglycemic activity of cultured yeast by oral administration in streptozotocin (STZ)-induced diabetic mice. Among 7 diluted fruit juice samples the diluted strawberry juice (1.74 g/L reducing sugar content) was chosen as the basal medium. After investigation of the effects of addition of various substances, including 1% of 5 different sugars and glycerol, 0.1% of 6 nitrogen-containing substances, and 1 ppm of 7 growth factors, the diluted strawberry juice added with 1% glucose, 0.1% yeast extract and 1 ppm aspartic acid was optimized at 20 °C with initial pH value of 6.0 for cultivating S. pastorianus no. 54 in flask. The scale-up system of a 5-L fermentor was further established by using the same medium with initial pH 6.0 and being incubated at 20 °C with an aeration rate of 1.2 vvm for 96 h. The hypoglycemic activity of yeast cells cultivated in fermentor was 3.11 times of that in flask. Oral administration of the cultured yeast at a dosage of 130 mg/kg body weight/day for 6 days could significantly reduce the plasma glucose content in STZ-induced diabetic mice and keep their body weights in the normal range. Copyright © 2011. Published by Elsevier B.V.

  15. Proliferation differentiation and therapeutic effect of short-term cultured murine bone marrow cells

    International Nuclear Information System (INIS)

    Zhao Zekun; Cong Jianbo

    1986-01-01

    Murine bone marrow cells were cultured in conditioned medium of muscle. After 24 hours of culture, both adherent and suspended cells appeared in the culture. The adherent cells mainly consisted of macrophages and the suspended cells were predominantly granulocytes. After 6 days, the total number of nucleated cells and CFU-C in the culture increased about 400% and 600% respectively, but CFU-S reduced to 21% approximately. Lymphocytes persisted only for 4 days. The stem cells (CFU-S) from 6-day culture were injected into the lethally irradiated syngenic mice. The 30 day survival rate of the treated mice was 89% whereas that of the controls was only 7%. The bone marrow cells in 2/8 of recipients sacrificed at 30 or 60 days were of donor type and 6/8 of the recipients were chimeras

  16. Triple co-culture cell model as an in vitro model for oral particulate vaccine systems

    DEFF Research Database (Denmark)

    Nielsen, Line Hagner; De Rossi, C.; Lehr, C-M.

    values of the co-cultures were found to be 860-1340 Ω∙cm2; the formulations were incubated with the co-cultures at this time point. From confocal microscopy images, it was observed that the THP-1 cells (macrophages) migrated into the overlying Caco-2 cell monolayer when the co-cultures were incubated......A triple co-culture cell model of Caco-2 cells, dendritic cells and macrophages (Figure 1) has previously been developed for studying intestinal permeability in a state of inflammation [1],[2]. The aim of this study was to investigate the applicability of this cell model for testing...... the model antigen ovalbumin was spray dried to obtain a particulate vaccine model system for testing in the cell model. The precursors were shown to form cubosomes when dispersed in aqueous medium, and was therefore used as the vaccine formulation for testing on the co-cultures. After 11 days, the TEER...

  17. Isolation and culture of pulmonary endothelial cells.

    Science.gov (United States)

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.

  18. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.

    2003-01-01

    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed...... to be simple duplicates for testing the effect of two induced factors-apical or basolateral addition of radioactive precursors and different apical media-on the incorporation of 14C-acetate and 32Pphosphate intotissue lipids. Unfortunately, they did not altogether give the same result. By accepting this fact...... and introducing the observed difference between batches as one of the factors in an expanded three-dimensional ANOVA, we were able to overcome an otherwisecrucial lack of sufficiently reproducible duplicate values. We could thereby show that the effect of changing the apical medium was much more marked when...

  19. Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

    International Nuclear Information System (INIS)

    Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

    1986-01-01

    Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with 3 H-thymidine. Cachetin treatment (10 -6 to 10 -10 M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and 3 H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (∼ 50%) at 10 -10 M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation

  20. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Seagrave, J.C.; Valdez, J.G.; Tobey, R.A.; Gurley, L.R.

    1985-06-01

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC1 2 , one being increased in amount while the other was reduced to an insignificant level. In ZnC1 2 -treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC1 2 -treated cells. While ZnC1 2 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC1 2 -treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC1 2 -treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  1. Study on recycling of waste rubbers as medium components for hydroponic culture of rose

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin-Kuk; Lee, Hyung-Gyu; Jeong, Byoung-Ryong; Hwang, Seung-Jae [Gyeongsang National Univ., Kumi(Korea)

    2000-06-30

    Recently, the efficient disposal of the waste rubber is necessary due to increasing amount of the waste rubbers. In this paper, method of recycling waste rubbers as components of medium for hydroponic rose culture was suggested. We investigated growth of rose, and macro- and micro-elements, pH and EC of the media amended with waste rubber. In the beginning of culture, stress symptoms such as thin brittle stem and incipient wilting were observed, but they disappeared in a few weeks. Concentration of Zn{sup 2+} in media at flowering increased in proportion to contents of waste tire in the media. pH of media at flowering were in the range of 5.70 to 6.35. Rose growth in all media, except in waste rock wool mixed with EPDM powder at 9:3 ratio, was normal and equivalent to the control in terms of stem length, number of stems harvested and fresh weight. (author). 10 refs., 5 tabs., 4 figs.

  2. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

    Science.gov (United States)

    Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

    2016-11-01

    To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

  3. Growth of Candida species in liquid culture medium for Trichomonas vaginalis.

    OpenAIRE

    Erdman, Y J; Holton, J M; Becker, A

    1984-01-01

    The growth of Candida spp from vaginal specimens in Bushby's liquid medium for T vaginalis was compared with that on Sabouraud's agar medium, and isolation was significantly greater in Bushby's medium (p less than 0.001). Isolations missed (4.43%) in Bushby's medium probably represented vaginal carriage of small numbers of Candida spp.

  4. In vitro plant regeneration of two cucumber (Cucumis sativum L. genotypes: Effects of explant types and culture medium

    Directory of Open Access Journals (Sweden)

    Grozeva Stanislava

    2014-01-01

    Full Text Available The effect of different phytohormone concentrations on callusogenesis and organogenesis in two cucumber genotypes were studied. It was established that the rate of plant regeneration depends on genotype, explant type and culture medium. Hypocotyls were found to be more responsive than cotyledons in morphogenesis. In vitro planlet-regenerants have been obtained in hypocotyls explants on culture medium with 1.0 and 2.0 mgL-1 BA for cultivar Gergana and in 1.0 and 3.0 mgL-1K-line 15B. Induction of regeneration in cotyledons were established only in cultivar Gergana on culture medium supplemented with 3.0 mgL-1 BA and in combination of 0.5 mgL-1IAA.

  5. Comparison of dry medium culture plates for mesophilic aerobic bacteria in milk, ice cream, ham, and codfish fillet products.

    Science.gov (United States)

    Park, Junghyun; Kim, Myunghee

    2013-12-01

    This study was performed to compare the performance of Sanita-Kun dry medium culture plate with those of traditional culture medium and Petrifilm dry medium culture plate for the enumeration of the mesophilic aerobic bacteria in milk, ice cream, ham, and codfish fillet. Mesophilic aerobic bacteria were comparatively evaluated in milk, ice cream, ham, and codfish fillet using Sanita-Kun aerobic count (SAC), Petrifilm aerobic count (PAC), and traditional plate count agar (PCA) media. According to the results, all methods showed high correlations of 0.989~1.000 and no significant differences were observed for enumerating the mesophilic aerobic bacteria in the tested food products. SAC method was easier to perform and count colonies efficiently as compared to the PCA and PAC methods. Therefore, we concluded that the SAC method offers an acceptable alternative to the PCA and PAC methods for counting the mesophilic aerobic bacteria in milk, ice cream, ham, and codfish fillet products.

  6. Individualized medicine for renal cell carcinoma: establishment of primary cell line culture from surgical specimens.

    Science.gov (United States)

    Kim, Fernando J; Campagna, Adriano; Khandrika, Lakshmipathi; Koul, Sweaty; Byun, Seok-Soo; vanBokhoven, Adrie; Moore, Ernest E; Koul, Hari

    2008-10-01

    The lack of effective "in vivo" and "in vitro" models to predict success of pharmacological therapy for patients with renal cell carcinoma, as well as, the variety of cancer cell types demands the development of better experimental models to understand the pathophysiology of the disease and evaluate drug sensitivity in vitro. To develop primary renal cancer cell culture irrespective of tumor grade and tumor type, harvested from the patient's pathological specimen immediately after the laparoscopic radical nephrectomy to study potential "in vivo" pharmacological sensitivity. A total of 24 patients (17 males and 7 females). Mean age of 63.1+/-3.1 y.o. The mean size of the renal masses was 7.56+/-3.1 cm. Normal and pathological renal tissue was collected immediately after the specimen was extracted and submitted to enzymatic digestion for 16-24 hours. Clear cell carcinoma cells were selected through multiple passages in DMEM medium supplemented with glucose and antibiotics. Establishment of cell line culture from all the patients' specimens irrespective of tumor grade and tumor type was achieved successfully. In addition to the tumor cell line culture, normal parenchyma tissue yielded primary cell lines to allow testing the response of tumor types to various pharmacological therapeutic agents and toxicity of such treatments to healthy tissue. From the initial collection of the specimens obtained after the removal of the kidney to the development of cell lines took occurred in average 32+6 hrs. The cells in culture showed characteristics of epithelial cells; like expression on cytokeratin and were maintained in culture for more than 20 passages. The development of renal cancer cell cultures in vitro is labor intense but may yield a more realistic model to tailor pharmacological therapies and predict therapeutic success prior to "in vivo" application-a step in the direction of individualized medicine for RCC.

  7. Cell division in Escherichia coli cultures monitored at single cell resolution

    Directory of Open Access Journals (Sweden)

    Luidalepp Hannes

    2008-04-01

    Full Text Available Abstract Background A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate. Results We monitored the division of individual cells in Escherichia coli cultures during different growth phases. Our experiments are based on the dilution of green fluorescent protein (GFP upon cell division, monitored by flow cytometry. The results show that the vast majority of E. coli cells in exponentially growing cultures divided uniformly. In cultures that had been in stationary phase up to four days, no cell division was observed. However, upon dilution of stationary phase culture into fresh medium, two subpopulations of cells emerged: one that started dividing and another that did not. These populations were detectable by GFP dilution and displayed different side scatter parameters in flow cytometry. Further analysis showed that bacteria in the non-growing subpopulation were not dead, neither was the difference in growth capacity reducible to differences in stationary phase-specific gene expression since we observed uniform expression of several stress-related promoters. The presence of non-growing persisters, temporarily dormant bacteria that are tolerant to antibiotics, has previously been described within growing bacterial populations. Using the GFP dilution method combined with cell sorting, we showed that ampicillin lyses growing bacteria while non-growing bacteria retain viability and that some of them restart growth after the ampicillin is removed. Thus, our method enables persisters to be monitored even in liquid cultures of wild type strains in which persister formation has low frequency. Conclusion In principle, the approaches developed here could be used to detect differences in cell division in response to different environmental conditions and in cultures of unicellular

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

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

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

  9. Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    International Nuclear Information System (INIS)

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

  10. Henrietta Lacks, HeLa cells, and cell culture contamination.

    Science.gov (United States)

    Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

    2009-09-01

    Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line.

  11. [Design and fabrication of a microfluidic chip for the co-culture of three cell types].

    Science.gov (United States)

    Wang, Shuaichao; Ge, Yuqing; Wu, Lei; Guo, Hui; Yang, Shiping; Jin, Qinghui

    2017-02-25

    Here a microfluidic chip with 'micro-dam' and 'micro-gap' has been designed and fabricated. It could isolate different cells and flow of medium in each region. It was found that the chip could realize the cells co-culture and patterning of human lung adenocarcinoma cell (A549), human embryonic lung fibroblast (HLF-1) and human endothelial cells (HUVECs), respectively. After 72 hours of culture, three kinds of cells grew well. It provided a developing technical platform for cell related research.

  12. Single cell protein production of Chlorella sp. using food processing waste as a cultivation medium

    Science.gov (United States)

    Putri, D.; Ulhidayati, A.; Musthofa, I. A.; Wardani, A. K.

    2018-03-01

    The aim of this study was to investigate the effect of various food processing wastes on the production of single cell protein by Chlorella sp. Three various food processing wastes i.e. tofu waste, tempeh waste and cheese whey waste were used as cultivation medium for Chlorella sp. growth. Sea water was used as a control of cultivation medium. The addition of waste into cultivation medium was 10%, 20%, 30%, 40%, and 50%. The result showed that the highest yield of cell mass and protein content was found in 50% tofu waste cultivation medium was 47.8 × 106 cell/ml with protein content was 52.24%. The 50% tofu waste medium showed improved cell yield as nearly as 30% than tempeh waste medium. The yield of biomass and protein content when 30% tempeh waste was used as cultivation medium was 37.1 × 106 cell/ml and 52%, respectively. Thus, food processing waste especially tofu waste would be a promising candidate for cultivation medium for single cell production from Chlorella sp. Moreover, the utilization of waste can reduce environmental pollution and increase protein supply for food supplement or animal feed.

  13. 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

  14. Priming cells for their final destination: microenvironment controlled cell culture by a modular ECM-mimicking feeder film.

    Science.gov (United States)

    Barthes, Julien; Vrana, Nihal E; Özçelik, Hayriye; Gahoual, Rabah; François, Yannis N; Bacharouche, Jalal; Francius, Grégory; Hemmerlé, Joseph; Metz-Boutigue, Marie-Hélène; Schaaf, Pierre; Lavalle, Philippe

    2015-09-01

    Mammalian cell culture is the starting point in many research studies focusing on biomedical applications. However, researchers have little control over the standardized cell microenvironment parameters. Here a modular ECM-mimicking surface coating for cell culture environment is designed. This substrate is a new and versatile thin film obtained by spin-coating of concentrated gelatin crosslinked by transglutaminase. It can be modified with respect to the biochemical and biophysical needs of the final cell destination, i.e. it delivers loaded multi-growth factors and serum components and allows for cell culture in a serum-free culture medium. Also, a well-known cell behavior modulator, the substrate stiffness, is controlled exogenously by addition of nanoparticles. In addition to growth factors, antimicrobial agents such as natural peptides are added to the substrate for limiting the repeated addition of antimicrobial agents to the culture medium and to prevent the increase of resistant bacterial strains in the culture environment. Finally, this substrate contains simultaneously ECM components, growth factors, stiffening elements and antimicrobial agents. It provides a favorable microenvironment and sterile conditions. It is a free-of-maintenance system, as cells will grow without addition of serum or antimicrobial cocktails. This low cost and easy-to-use substrate could emerge as a new standard for cell culture.

  15. Kinetics of phototoxicity of Fischer's medium for L5178Y leukemic cells

    International Nuclear Information System (INIS)

    Griffin, F.M.; Ashland, G.; Capizzi, R.L.

    1981-01-01

    The uncontrolled exposure of Fischer's medium to cool white fluorescent (CWF) light or other sources emitting near-ultraviolet or visible light absorbance by riboflavin is a crucial random variable in experiments which utilize L5178Y cells and this medium. The radiation effects of CWF light result in the rapid development of toxic photoproducts in the medium which are cytostatic at lower doses of radiation and cytotoxic at higher doses. After a 24-hr suspension in medium irradiated for 3 or 48 hr, the cloning efficiencies of cells subsequently plated in light-protected medium were 87 and 3%, respectively. The corresponding near-ultraviolet doses for these periods of exposure to CWF light were 0.22 x 10(4) for a 3-hr exposure and 3.47 x 10(4) J/sq m for a 48-hr exposure. Cells incubated in lightly irradiated medium resumed growth at nearly normal rates following a 24- to 48-hr period in which no increase in cell numbers occurred. Exposure of medium containing riboflavin, but not tryptophan or tyrosine, to CWF light also produces toxic medium. Tryptophan enhances riboflavin-induced phototoxicity, whereas tyrosine diminishes this effect. As photosusceptibility of this system is very high, Fischer's medium must be fully protected from all sources of light absorbable by riboflavin

  16. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

    Science.gov (United States)

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

    2016-08-01

    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence

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

    Science.gov (United States)

    Takahashi, Eiji; Yamaoka, Yoshihisa

    2017-11-01

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

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

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

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

  19. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli.

    Science.gov (United States)

    Albiniak, Anna M; Matos, Cristina F R O; Branston, Steven D; Freedman, Robert B; Keshavarz-Moore, Eli; Robinson, Colin

    2013-08-01

    The twin-arginine translocation (Tat) system transports folded proteins across the plasma membrane in bacteria, and heterologous proteins can be exported by this pathway if a Tat-type signal peptide is present at the N-terminus. The system thus has potential for biopharmaceutical production in Escherichia coli, where export to the periplasm is often a favoured approach. Previous studies have shown that E. coli cells can export high levels of protein by the Tat pathway, and the protein product accummulates almost exclusively in the periplasm. In this study, we analysed E. coli cells that express the Bacillus subtilis TatAdCd system in place of the native TatABC system. We show that a heterologous model protein, comprising the TorA signal peptide linked to green fluorescent protein (TorA-GFP), is efficiently exported by the TatAdCd system. However, whereas the GFP is exported initially to the periplasm during batch fermentation, the mature protein is increasingly found in the extracellular culture medium. By the end of a 16-h fermentation, ~ 90% of exported GFP is present in the medium as active mature protein. The total protein profiles of the medium and periplasm are essentially identical, confirming that the outer membrane becomes leaky during the fermentation process. The cells are otherwise intact, and there is no large-scale release of cytoplasmic contents. Export levels are relatively high, with ~ 0.35 g GFP·L⁻¹ culture present in the medium. This system thus offers a means of producing recombinant protein in E. coli and harvesting directly from the medium, with potential advantages in terms of ease of purification and downstream processing. © 2013 FEBS.

  20. Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells.

    Science.gov (United States)

    Kim, Narae; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2017-01-01

    One of the parameters that greatly affects homeostasis in the body is the pH. Regarding reproductive biology, germ cells, such as oocytes or sperm, are exposed to severe changes in pH, resulting in dramatic changes in their characteristics. To date, the effect of the pH has not been investigated regarding the reprogramming of somatic cells and the maintenance and differentiation of pluripotent stem cells. In order to investigate the effects of the pH on cell culture, the methods to produce induced pluripotent stem cells (iPSCs) and to differentiate embryonic stem cells (ESCs) into mesendoderm and neuroectoderm were performed at each medium pH from 6.6 to 7.8. Using the cells of the Oct4 -GFP (green fluorescent protein) carrying mouse, the effects of pH changes were examined on the timing and colony formation at cell reprogramming and on the cell morphology and direction of the differentiation of the ESCs. The colony formation rate and timing of the reprogramming of the somatic cells varied depending on the pH of the culture medium. In addition, mesendodermal differentiation of the mouse ESCs was enhanced at the high pH level of 7.8. These results suggest that the pH in the culture medium is one of the key factors in the induction of the reprogramming of somatic cells and in the differentiation of pluripotent stem cells.

  1. Degradation of high density lipoprotein in cultured rat luteal cells

    International Nuclear Information System (INIS)

    Rajan, V.P.; Menon, K.M.J.

    1986-01-01

    In rat ovary luteal cells, degradation of high density lipoprotein (HDL) to tricholoracetic acid (TCA)-soluble products accounts for only a fraction of the HDL-derived cholesterol used for steroidogenesis. In this study the authors have investigated the fate of 125 I]HDL bound to cultured luteal cells using pulse-chase technique. Luteal cell cultures were pulse labeled with [ 125 I]HDL 3 and reincubated in the absence of HDL. By 24 h about 50% of the initallay bound radioactivity was released into the medium, of which 60-65% could be precipitated with 10% TCA. Gel filtration of the chase incubation medium on 10% agarose showed that the amount of TCA-soluble radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity eluted over a wide range of molecular weights (15,000-80,000), and there was very little intact HDL present. Electrophoresis of the chase medium showed that component of the TCA-precipitable portion had mobility similar to apo AI. Lysosomal inhibitors of receptor-mediated endocytosis had no effect on the composition or quantity of radioactivity released during chase incubation. The results show that HDL 3 binding to luteal cells is followed by complete degradation of the lipoprotein, although the TCA-soluble part does not reflect the extent of degradation

  2. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    Science.gov (United States)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  3. Algal autolysate medium to label proteins for NMR in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia [University of Florence, Magnetic Resonance Center (CERM) (Italy); Neri, Sara [Giotto Biotech S.R.L. (Italy); Fragai, Marco, E-mail: fragai@cerm.unifi.it [University of Florence, Magnetic Resonance Center (CERM) (Italy)

    2016-04-15

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in {sup 15}N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained.

  4. Effect of the temperature and the culture medium on the productivity of two generations of dihybrid crosses in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Alejandro Balbín

    2000-01-01

    Full Text Available In the present study we analize the productivity of the dihybrid cross +//+ e//e x vg//vg +//+ and its reciprocal, carried on up to the second generation under different environmental conditions: two culture media (maize and banana and two temperatures (23 and 26ºC. The differences were shown to be statistically significantfor the temperature in the first generation, the culture medium and the parental line in the second generation. At a general level, the significant factors were the temperature and the culture medium. Perhaps the genotipic effects caused by the parental line are balanced and masked, when considered at this level. The temperature of 26ºC and the culture media of maize showed always the best productivity.

  5. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures.

    Science.gov (United States)

    Kraus, Armin; Täger, Joachim; Kohler, Konrad; Manoli, Theodora; Haerle, Max; Werdin, Frank; Hoffmann, Jürgen; Schaller, Hans-Eberhard; Sinis, Nektarios

    2010-01-01

    The efficacy of Schwann-cell cultivation can be enhanced by in vitro predegeneration of the harvested cells compared to immediate culture. The aim of this study was to improve Schwann-cell culture efficacy by comparing three different durations of predegeneration. The sciatic and median nerves of 6-8-week-old Lewis rats were harvested and subjected to either 2-day, 7-day, or 14-day predegeneration in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin. Afterward, tissue was enzymatically dissociated and placed in a modified melanocyte growth medium. The cell count was determined immediately after dissociation while the cell purity was determined one subculture/trypsinization cycle later after cell attachment to the culture plate by means of optical microscopy and immunocytochemistry. Particular attention was then paid to the Schwann-cell-to-fibroblast relation. The cumulative cell count in the culture was 5.8 x 10(5) for 2-day, 1.12 x 10(6) for 7-day, and 1.48 x 10(6) for 14-day predegeneration. The culture purity was approximately equal for 2- and 7-day predegeneration (88% Schwann cells, 12% fibroblasts after 2 days; 85% Schwann cells, 15% fibroblasts after 7 days). After 14 days, however, cell cultures were significantly debased by fibroblast proliferation (57% Schwann cells, 43% fibroblasts). In vitro predegeneration is a particularly suitable procedural method to increase the cultural Schwann-cell yield. The number of cultivated rat Schwann cells is doubled by 7-day in vitro predegeneration in comparison to 2-day predegeneration. After 14-day predegeneration, however, the culture is significantly debased by fibroblasts. Therefore, 7-day in vitro predegeneration is an advisable predegeneration period.

  6. Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

    2010-10-15

    NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

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

    Science.gov (United States)

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

    1992-12-05

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

  8. Adipose-derived stem cell conditioned medium attenuates cisplatin-triggered apoptosis in tongue squamous cell carcinoma.

    Science.gov (United States)

    Chiu, Yu-Jen; Yang, Jai-Sing; Hsu, Han-Shui; Tsai, Chi-Han; Ma, Hsu

    2018-02-01

    Autologous fat grafting procedures have noted a markedly increased frequency, not only for cosmetic purposes, but also for deformities after head and neck cancer and breast cancer surgery. Carcinogenesis is always a major concern in cell therapy-related issues. However, there is no literature discussing this issue in head and neck squamous cell carcinoma patients. To evaluate the interaction of tongue cancer cells and adipose-derived stem cells, we performed a series of in vitro experiments. Our results demonstrated that cisplatin significantly reduced the viabilities of SCC‑25 and CAL‑27 cells in a concentration-dependent manner, but it had low cytotoxicity in cisplatin-resistant CAL‑27 (CAR) cells. There was no significant difference in terms of viability among the SCC‑25, CAL‑27, and CAR cells in the adipose-derived stem cell conditioned medium and control groups. There was also no significant difference in terms of cell migration as determined by wound healing assay of SCC‑25, CAL‑27, and CAR cells between the adipose-derived stem cell conditioned medium treatment and control treatment. Importantly, the adipose-derived stem cell conditioned medium attenuated cisplatin-triggered cell death in the SCC‑25 and CAL‑27 cells. Moreover, adipose-derived stem cell conditioned medium markedly inhibited cisplatin-induced apoptotic cell death (sub‑G1 phase) in the CAL‑27 cells. Western blot analyses indicated that cisplatin-induced reductions in pro‑caspase‑3, pro‑caspase‑9, phospho-BAD, phospho-IGF-1R, phospho-AKT, and phospho-ERK in CAL‑27 cells were reversed by adipose-derived stem cell conditioned medium supplement. Taken together, we provide evidence that adipose-derived stem cell conditioned medium protects CAL‑27 cells from cisplatin-induced cell death, possibly through upregulation of the IGF-1R/AKT/ERK signaling pathway.

  9. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  10. The influence of the type of embryo culture medium on neonatal birthweight after single embryo transfer in IVF.

    Science.gov (United States)

    Vergouw, Carlijn G; Kostelijk, E Hanna; Doejaaren, Els; Hompes, Peter G A; Lambalk, Cornelis B; Schats, Roel

    2012-09-01

    Does the type of medium used to culture fresh and frozen-thawed embryos influence neonatal birthweight after single embryo transfer (SET) in IVF? A comparison of two commercially available culture media showed no significant influence on mean birthweight and mean birthweight adjusted for gestational age, gender and parity (z-scores) of singletons born after a fresh or frozen-thawed SET. Furthermore, we show that embryo freezing and thawing cycles may lead to a significantly higher mean birthweight. Animal studies have shown that culture media constituents are responsible for changes in birthweight of offspring. In human IVF, there is still little knowledge of the effect of medium type on birthweight. Until now, only a small number of commercially available culture media have been investigated (Vitrolife, Cook(®) Medical and IVF online medium). Our study adds new information: it has a larger population of singleton births compared with the previously published studies, it includes outcomes of other media types (HTF and Sage(®)), not previously analysed, and it includes data on frozen-thawed SETs. This study was a retrospective analysis of birthweights of singleton newborns after fresh (Day 3) or frozen-thawed (Day 5) SET cycles, using embryos cultured in either of two different types of commercially available culture media, between 2008 and 2011. Before January 2009, a single-step culture medium was used: human tubal fluid (HTF) with 4 mg/ml human serum albumin. From January 2009 onwards, a commercially available sequential medium was introduced: Sage(®), Quinn's advantage protein plus medium. Singletons born after a fresh SET (99 embryos cultured in HTF and 259 in Sage(®)) and singletons born after a frozen-thawed SET (32 embryos cultured in HTF only, 41 in HTF and Sage(®) and 86 in Sage(®) only) were analysed. Only patients using autologous gametes without the use of a gestational carrier were considered. Also excluded were (vanishing) twins, triplets

  11. Cell Culture on MEMS Platforms: A Review

    Science.gov (United States)

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  12. New plant-growth medium for increased power output of the Plant-Microbial Fuel Cell

    NARCIS (Netherlands)

    Helder, M.; Strik, D.P.B.T.B.; Hamelers, H.V.M.; Kuijken, R.C.P.; Buisman, C.J.N.

    2012-01-01

    In a Plant-Microbial Fuel Cell anode-conditions must be created that are favorable for plant growth and electricity production. One of the major aspects in this is the composition of the plant-growth medium. Hoagland medium has been used until now, with added phosphate buffer to reduce potential

  13. Organisational culture as a part in the development of open innovation - the perspective of small and medium-sized enterprises

    Directory of Open Access Journals (Sweden)

    Szymańska Katarzyna

    2016-05-01

    Full Text Available The ability to introduce various concepts and business models is nowadays a prerequisite of creating a competitive advantage. This is to a large extent closely linked to the ability of enterprises to create, implement and disseminate a variety of innovative solutions. Today the use of open innovation is a necessity. This applies not only to large organisations, but also to small and medium-sized enterprises. In order to implement open innovation, small and medium-sized enterprises need to effectively manage their own growth through the preparation of appropriate strategies and the development of a model that encompasses all changes, taking into account a number of factors related to the growth dynamics of this sector. It is understood that an appropriate organisational culture plays an important role in the implementation of innovation in the sector of small and medium-sized enterprises. There are many indications that a cultural mismatch and misunderstanding are the main reasons for major problems related to the low level of implementation of innovation by small and medium-sized enterprises. The aim of the paper is to outline the issue of the impact of organisational culture on the development of the concept of open innovation in the sector of small and medium-sized enterprises.

  14. Growth and development of rabbit oocytes in vitro: effect of fetal bovine serum concentration on culture medium.

    Science.gov (United States)

    Sugimoto, H; Kida, Y; Miyamoto, Y; Kitada, K; Matsumoto, K; Saeki, K; Taniguchi, T; Hosoi, Y

    2012-09-15

    The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P medium for IVG improved developmental competence of rabbit oocytes grown in vitro. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir ( Pseudotsuga menziesii) shoot cultures.

    Science.gov (United States)

    Chen, Chien-Chih; Bates, Rick; Carlson, John

    2014-01-01

    The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change on explant growth performance and 3) assess the effects of adding a pH stabilizer, 2-(N-morpholino)ethanesulfonic acid (MES) that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth.

  16. Children’s picturebook on sexual educationas a cultural and political medium

    Directory of Open Access Journals (Sweden)

    Małgorzata Cackowska

    2011-11-01

    Full Text Available In my article I deal with a social construction of meanings of picturebooks’ content and form in Poland and abroad, so also with what kinds of discourses and ideologies determine the conditions of picturebooks’ production in societies under analysis. For the analysis I have chosen picturebooks which deal with sexual education. The methodology applied in the research consists mostly of content analysis and critical discourse analysis. The research is a part of a bigger collaborative project called “Discursive construction of subjectivity” financed by Ministry of Science and Higher Education in Poland, grant no. N 10702632/3637, and conducted at the University of Gdansk. I present, basing on the empirical material, the critique of the dominant discourse in Poland which is powerful in the production of picturebooks, which is based on the conservative ideology and social and sexual roles defined in stereotypical, hierarchical and heterosexual terms. In this aura discourses based on liberal or radical ideologies are marginalized.The results show the knowledge/power relations, symptoms of symbolic violence in exemplified discourses and explain to what practices of ideological and political control the subject is exposed. In this context a picturebook is seen as a meaningful cultural and political medium, within the content and form of which various (possible ideologies and conceptions of the child are included to or excluded from social environment, what can occur as a real issue for educational theory and practice.

  17. Effects of cultural medium on the formation and antitumor activity of polysaccharides by Cordyceps gunnii.

    Science.gov (United States)

    Zhu, Zhen-Yuan; Liu, Xiao-Cui; Tang, Ya-Li; Dong, Feng-Ying; Sun, Hui-Qing; Chen, Lu; Zhang, Yong-Min

    2016-10-01

    The effects of culture medium composition (i.e., carbon and nitrogen sources) on the growth of mycelia, molecular weight distribution and antitumor activity of intracellular polysaccharides (IPS) from Cordyceps gunnii were investigated. Sucrose and peptone were proved to be the best carbon and nitrogen sources for mycelia growth and remarkably improved IPS production. When the sucrose concentration was 2.0%, the mycelium yield reached up to 15.94±1.26 g/L, but with lower IPS yield; whereas the sucrose concentration was 4.5%, IPS yield reached to a maximum of 138.78±3.89 mg/100 mL. The effects of different carbon/nitrogen (C/N) ratios with equal amounts of carbon source matter on the mycelia and IPS formation were optimized. It found that the yield of mycelia and IPS were both reached to the highest at a C/N ratio of 10:3. In addition, the IPS had the highest macro molecular polysaccharide content and antitumor activity when sucrose concentration was 3.5% and the C/N ratio was 10:1.5. Thus, there was a positive correlation between molecular weight distribution and antitumor activity of IPS by C. gunnii. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Optimization of a Culture Medium Using the Taguchi Approach for the Production of Microorganisms Active in Odorous Compound Removal

    Directory of Open Access Journals (Sweden)

    Krzysztof Makowski

    2017-07-01

    Full Text Available The aim of this work was to develop the composition of a medium for the cultivation of six microbial strains forming a deodorizing consortium: Pseudomonas fluorescens, Enterococcus faecium, Bacillus subtilis, Bacillus megaterium, Leuconostoc mesenteroides and Lactobacillus plantarum. The study focused on the optimization of a highly efficient culture medium composed of readily available components of plant origin to maximize microbial biomass yields, and to create a less expensive alternative to the commercial Tryptic Soy Broth medium (TSB. After preliminary efficiency screening of all tested media components, we selected four substrates for further optimization—soy protein concentrate (SPC, glucose or sucrose, and phosphate salts. The final concentrations of all components were fine-tuned using the Taguchi design for experiments according to an L9 array. Taguchi optimization led to formulation of a culture medium, which was approximately 5 times cheaper than TSB (depending on the components used. Consequently, microbial biomass yields were improved by up to 15-fold (1564%, depending on the strain. The results obtained in the laboratory experiments were then confirmed in pilot- (42 L and industrial- (300 L scale fermentation. Our results show that this method of using a parallel culture microbioreactor with the Taguchi approach can be recommended for optimization of culture media based on substrates of plant origin.

  19. Effects of radiation on cultured fish cells

    International Nuclear Information System (INIS)

    Etoh, Hisami; Suyama, Ippei

    1980-01-01

    A new fibroblastic cell line was established in our laboratory from the caudal fin of the goldfish, C. auratus. The cells, designated CAF, have been subcultured over 80 passages since initiation in August, 1977. A brief description of cell cultivation and colony formation is presented. The plating efficiency obtained was considerably higher than those reported for other fish cell lines. CAF cells were irradiated with 250, 500, 1,000, 2,000, and 3,000 R of x-rays at a dose rate of 80 R/min in air. The survival parameters changed when the number of passages of culture increased. Values for D 0 , D sub(q), and n obtained from cells irradiated at the 70th passage were calculated to be 650 R, 700 R, and 2.7 respectively. Thus CAF cells would be several times as resistant in general as cultured mammalian cells. The cells irradiated with 1,000 R of x-rays received a second dose from 250 to 2,000 R at intervals of 3, 6, and 24 hr. The cells kept at 26 0 C showed a pronounced recovery from sublethal damage during the intervals between two doses. Magnitude of recovery was larger if the interval was longer under the present experimental conditions. These results may indicate that the recovery observed at an individual level accounts partly for that in vitro. (author)

  20. Cat and Dog Primordial Follicles Enclosed in Ovarian Cortex Sustain Viability after In vitro Culture on Agarose Gel in a Protein-Free Medium

    Science.gov (United States)

    Fujihara, M; Comizzoli, P; Wildt, DE; Songsasen, N

    2014-01-01

    Contents Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α-minimum essential medium (α-MEM) or MEM supplemented with 10% fetal bovine serum (FBS), 10% knock-out serum replacement (KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α-MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α-MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p cat follicle viability, whereas the latter was superior (p dog follicle survival. Likewise, dog follicle viability was enhanced (p cat, the agarose gel better (p cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange. PMID:23279476

  1. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    state, may be of significant benefit for cfDNA research. Methods CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose...... environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.......Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...

  2. Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

    Science.gov (United States)

    Jiang, Shan; Liu, Mei; Wang, Baojie; Jiang, Keyong; Wang, Lei

    2011-11-01

    Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

  3. IVF culture medium affects human intrauterine growth as early as the second trimester of pregnancy.

    Science.gov (United States)

    Nelissen, Ewka C M; Van Montfoort, Aafke P A; Smits, Luc J M; Menheere, Paul P C A; Evers, Johannes L H; Coonen, Edith; Derhaag, Josien G; Peeters, Louis L; Coumans, Audrey B; Dumoulin, John C M

    2013-08-01

    When does a difference in human intrauterine growth of singletons conceived after IVF and embryo culture in two different culture media appear? Differences in fetal development after culture of embryos in one of two IVF media were apparent as early as the second trimester of pregnancy. Abnormal fetal growth patterns are a major risk factor for the development of chronic diseases in adult life. Previously, we have shown that the medium used for culturing embryos during the first few days after fertilization significantly affects the birthweight of the resulting human singletons. The exact onset of this growth difference was unknown. In this retrospective cohort study, all 294 singleton live births after fresh embryo transfer in the period July 2003 to December 2006 were included. These embryos originated from IVF treatments that were part of a previously described clinical trial. Embryos were allocated to culture in either Vitrolife or Cook commercially available sequential culture media. We analysed ultrasound examinations at 8 (n = 290), 12 (n = 83) and 20 weeks' (n = 206) gestation and used first-trimester serum markers [pregnancy-associated plasma protein-A (PAPP-A) and free β-hCG]. Differences between study groups were tested by the Student's t-test, χ(2) test or Fisher's exact test, and linear multivariable regression analysis to adjust for possible confounders (for example, parity, gestational age at the time of ultrasound and fetal gender). A total of 294 singleton pregnancies (Vitrolife group nVL = 168, Cook group: nC = 126) from 294 couples were included. At 8 weeks' gestation, there was no difference between crown-rump length-based and ovum retrieval-based gestational age (ΔGA) (nVL = 163, nC = 122, adjusted mean difference, -0.04 days, P = 0.84). A total of 83 women underwent first-trimester screening at 12 weeks' gestation (nVL = 45, nC = 38). ΔGA, nuchal translucency (multiples of median, MoM) and PAPP-A (MoM) did not differ between the study

  4. Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing.

    Science.gov (United States)

    Pfannebecker, Jens; Schiffer-Hetz, Claudia; Fröhlich, Jürgen; Becker, Barbara

    2016-11-01

    In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (a w )-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS ® parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1-0.2% ammonium salts ((NH 4 ) 2 HPO 4 , (NH 4 ) 2 SO 4 or NH 4 NO 3 ), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 10 2 CFU/g within a time period of 2-3days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124h in YEG50, 95.5h in MYG50 and 55h in OM bouillon. Compared to YEG50 the maximum CO 2 transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3days for Z. rouxii and T. delbrueckii

  5. Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts.

    Science.gov (United States)

    Kim, H-S; Son, H-Y; Kim, S; Lee, G-S; Park, C-H; Kang, S-K; Lee, B-C; Hwang, W-S; Lee, C-K

    2007-02-01

    The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.

  6. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture...

  7. Method and Apparatus for a Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Kleis, Stanley J. (Inventor); Geffert, Sandra K. (Inventor); Gonda, Steve R. (Inventor)

    2015-01-01

    A bioreactor and method that permits continuous and simultaneous short, moderate, or long term cell culturing of one or more cell types or tissue in a laminar flow configuration is disclosed, where the bioreactor supports at least two laminar flow zones, which are isolated by laminar flow without the need for physical barriers between the zones. The bioreactors of this invention are ideally suited for studying short, moderate and long term studies of cell cultures and the response of cell cultures to one or more stressors such as pharmaceuticals, hypoxia, pathogens, or any other stressor. The bioreactors of this invention are also ideally suited for short, moderate or long term cell culturing with periodic cell harvesting and/or medium processing for secreted cellular components.

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

    Directory of Open Access Journals (Sweden)

    Mohammad Serajur RAHMAN

    2012-02-01

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

  9. Embryo forming cells in carrot suspension cultures

    NARCIS (Netherlands)

    Toonen, M.A.J.

    1997-01-01


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

  10. 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

  11. Producing biodiesel from cotton seed oil using Rhizopus oryzae ATTC #34612 whole cell biocatalysts: Culture media and cultivation period optimization

    Science.gov (United States)

    The effect of culture medium composition and cultivation time on biodiesel production by Rhizopus oryzae ATCC #34612 whole cell catalysts, immobilized on novel rigid polyethylene biomass supports, was investigated. Supplementation of the medium with carbon sources led to higher lipase activity and i...

  12. Optimization of Culture Medium for the Growth of Candida sp. and Blastobotrys sp. as Starter Culture in Fermentation of Cocoa Beans (Theobroma cacao) Using Response Surface Methodology (RSM).

    Science.gov (United States)

    Mahazar, N H; Zakuan, Z; Norhayati, H; MeorHussin, A S; Rukayadi, Y

    2017-01-01

    Inoculation of starter culture in cocoa bean fermentation produces consistent, predictable and high quality of fermented cocoa beans. It is important to produce healthy inoculum in cocoa bean fermentation for better fermented products. Inoculum could minimize the length of the lag phase in fermentation. The purpose of this study was to optimize the component of culture medium for the maximum cultivation of Candida sp. and Blastobotrys sp. Molasses and yeast extract were chosen as medium composition and Response Surface Methodology (RSM) was then employed to optimize the molasses and yeast extract. Maximum growth of Candida sp. (7.63 log CFU mL-1) and Blastobotrys sp. (8.30 log CFU mL-1) were obtained from the fermentation. Optimum culture media for the growth of Candida sp., consist of 10% (w/v) molasses and 2% (w/v) yeast extract, while for Blastobotrys sp., were 1.94% (w/v) molasses and 2% (w/v) yeast extract. This study shows that culture medium consists of molasses and yeast extract were able to produce maximum growth of Candida sp. and Blastobotrys sp., as a starter culture for cocoa bean fermentation.

  13. Type of cell death induced by seven metals in cultured mouse osteoblastic cells.

    Science.gov (United States)

    Contreras, René García; Vilchis, José Rogelio Scougall; Sakagami, Hiroshi; Nakamura, Yuko; Nakamura, Yukio; Hibino, Yasushi; Nakajima, Hiroshi; Shimada, Jun

    2010-01-01

    The use of dental metal alloys in the daily clinic makes it necessary to evaluate the cytotoxicity of eluted metal components against oral cells. However, the cytotoxic mechanism and the type of cell death induced by dental metals in osteoblasts have not been well characterized. This study investigated the cytotoxicity of seven metals against the mouse osteoblastic cell line MC3T3-E1. alpha-MEM was used as a culture medium, since this medium provided much superior proliferation of MC3T3-E1 cells over DMEM. Ag (NH(3))(2)F was the most cytotoxic, followed by CuCl>CuCl(2) >CoCl(2), NiCl(2)>FeCl(3) and FeCl(2) (least toxic). None of the metals showed any apparent growth stimulating effect (so-called 'hormesis') at lower concentrations. A time course study demonstrated that two hours of contact between oral cells and Ag (NH(3))(2)F, CuCl, CoCl(2) or NiCl(2) induced irreversible cell death. Contact with these metals induced a smear pattern of DNA fragmentation without activation of caspase-3. Preincubation of MC3T3-E1 cells with either a caspase inhibitor (Z-VAD-FMK) or autophagy inhibitors (3-methyladenine, bafilomycin) failed to rescue them from metal cytotoxicity. These data suggest the induction of necrotic cell death rather than apoptosis and autophagy by metals in this osteoblastic cell line.

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

    Science.gov (United States)

    Ashmore, S E; Shapcott, A S

    1989-08-01

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

  15. Production of Mushroom Mycelium as a Protein and Fat Source in Submerged Culture in Medium of Vinasse

    Science.gov (United States)

    Falanghe, H.

    1962-01-01

    Of ten mushroom cultures investigated, only Agaricus campestris, Boletus indecisus, and Tricholoma nudum were capable of growing in submerged culture in medium of vinasse with added salts. Higher fermentative efficiencies were found under these conditions than in medium containing molasses or waste sulfite liquor. A. campestris showed a better capacity to produce protein but, since B. indecisus is capable of developing greater mycelium weight, its fermentative efficiencies are comparable. Both microorganisms could be grown in medium of vinasse with greatly varied amounts, producing higher mycelial weight in media with greater vinasse. The capacity of B. indecisus and A. campestris to utilize the noncarbohydrate fraction in total solids, instead of the total carbohydrates when they are in smaller amount, was observed in medium containing vinasse. B. indecisus and A. campestris were easily separated by filtration from the medium, although T. nudum was difficult to separate by this procedure. In experiments with A. campestris, the adaptative capacity of the organism to vinasse was demonstrated. PMID:13962715

  16. 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

  17. Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures.

    Science.gov (United States)

    Gonzales, Veronica K; de Mulder, Eric L W; de Boer, Trix; Hannink, Gerjon; van Tienen, Tony G; van Heerde, Waander L; Buma, Pieter

    2013-11-01

    Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three healthy adult donors. Human meniscal fibrochondrocytes (MFCs) were isolated from resected tissue after a partial meniscectomy on a young patient. Passage-4 MFCs were cultured in monolayer for 24 h, and 3 and 7 days. Six different culture media were used containing different amounts of either PRP or PPP and compared to a medium containing 10% FBS. dsDNA was quantified, and gene expression levels of collagen types I and II and aggrecan were measured at different time points with quantitative polymerase chain reaction in the cultured MFCs. After 7 days, the dsDNA quantity was significantly higher in MFCs cultured in 10% and 20% PRP compared to the other PRP and PPP conditions, but equal to 10% FBS. Collagen type I expression was lower in MFCs cultured with medium containing 5% PRP, 10% and 20% PPP compared to FBS. When medium with 10% PRP or 20% PRP was used, expressions were not significantly different from medium containing 10% FBS. Collagen type II expression was absent in all medium conditions. Aggrecan expression did not show differences between the different media used. However, after 7 days a higher aggrecan expression was measured in most culture conditions, except for 5% PRP, which was similar compared to FBS. Statistical significance was found between donors at various time points in DNA quantification and gene expression, but the same donors were not statistically different in all conditions. At 7 days cell cultured with 10% PRP and 20% PRP showed a higher density, with large areas of clusters, compared to other conditions. In an MFC culture medium, FBS can be replaced by 10% PRP or 20% PRP without altering proliferation and gene expression of human MFCs.

  18. EFFECT OF MACROLIDE ANTIBIOTICS ON VARIOUS CELL CULTURES IN VITRO: 2. CELL BIOCHEMISTRY

    Directory of Open Access Journals (Sweden)

    Anton Kováčik

    2012-12-01

    Full Text Available he aim of our study was to evaluate the effect of macrolide antibiotics (tilmicosin, tylosin and spiramycin on the cellular biochemistry using different cell cultures in vitro. Cellular lines from animal tissues (VERO cells - kidney cells of Macacus Rhesus, FE cells - feline embryonal cells and BHK21 - cellular line from young hamster kidneys were used. The effect was assessed after 24 hours of culture. We studied the concentration of calcium (Ca, magnesium (Mg, sodium (Na, potassium (K, chlorides (Cl, total proteins (TP and cholesterol (Chol. Biochemical analysis of BHK21 cells cultivated with tilmicosin showed a significant decrease in the concentration of Ca, Cl and TP in almost all experimental groups. No significant differences were found in the FE cells. The highest concentrations of tilmicosin led to a significant increase of all analyzed elements and TP in medium in the VERO cells. The effect of tylosin on the BHK21 cell metabolism showed a significant decrease in the concentration of Na and Cl in the all experimental groups and a significant decrease in the concentration of TP in the groups to which more than 700 µg.ml-1 was added. No significant differences were found in the FE and VERO cells. Biochemical analysis of BHK21 cells with spyramicin showed a significant decrease in the concentration of Na in the all experimental groups and a significant decrease in the concentration of Cl and TP in the cell cultures with 100 µg.ml-1, 150 µg.ml-1, 200 µg.ml-1, 300 µg.ml-1 concentrations of spyramycin. The highest concentrations of spyramycin caused a significant increase of Na and a significant decrease of Chol in the FE cells. No significant differences were found in the VERO cells except increased total proteins at the highest concentration of spyramycin.

  19. Plasticity of mesenchymal stem cells from mouse bone marrow in the presence of conditioned medium of the facial nerve and fibroblast growth factor-2.

    Science.gov (United States)

    Lucena, Eudes Euler de Souza; Guzen, Fausto Pierdoná; Cavalcanti, José Rodolfo Lopes de Paiva; Marinho, Maria Jocileide de Medeiros; Pereira, Wogelsanger Oliveira; Barboza, Carlos Augusto Galvão; Costa, Miriam Stela Mariz de Oliveira; do Nascimento Júnior, Expedito Silva; Cavalcante, Jeferson Sousa

    2014-01-01

    A number of evidences show the influence of the growth of injured nerve fibers in peripheral nervous system as well as potential implant stem cells (SCs). The SCs implementation in the clinical field is promising and the understanding of proliferation and differentiation is essential. This study aimed to evaluate the plasticity of mesenchymal SCs from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants and fibroblast growth factor-2 (FGF-2). The growth and morphology were assessed for over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for glial fibrillary acidic protein (GFAP), protein OX-42 (OX-42), protein associated with microtubule MAP-2 (MAP-2), protein β-tubulin III (β-tubulin III), neuronal nuclear protein (NeuN), and neurofilament 200 (NF-200). Cells cultured with conditioned medium alone or combined with FGF-2 showed morphological features apparently similar at certain times to neurons and glia and a significant proliferative activity in groups 2 and 4. Cells cultivated only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN, and NF-200. This study improves our understanding of the plasticity of mesenchymal cells and allows the search for better techniques with SCs.

  20. Using CR1aa versus KSOM as the culture medium for in vitro embryo production of cattle

    Directory of Open Access Journals (Sweden)

    Endang Triwulaninngsih

    2002-03-01

    Full Text Available This research has been conducted at the laboratory of in vitro fertilization in the Department of Animal Science University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. Oocytes were matured in TCM- 199 medium (in 5% CO2 incubator and at 390C enriched with follicle stimulating hormone (FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10% Fetal Calf Serum (FCS. The oocytes were fertilized in vitro with motile sperm and incubation between sperm and oocytes in fertilization medium Tyroide Albumin Lactate Pyruvate (TALP for 20 hours. All zygotes were cultured in CR1aa (n=1549 medium versus modification of protein-free pottasium simplex optimized medium (KSOM (n=675 up to blastocyst stage and were fed FCS 5 μl/50 μl medium on day 6, as treatment A and B respectively. Data were analyzed by completely randomized design with SAS program. Percentages of cleavage, morula, blastocyst, expanded blastocyst, unfertilized and degenerated ova in this study were 91.4% vs 75.6 %; 75.6% vs 58.9%; 61.5% vs 38.5%; 31.2% vs 5.1%, 8.6% vs 24.4%, 15.7% vs 8% which were significantly different (P<0.01 for treatment CR1aa and KSOM respectively. Based on this study, CR1aa medium is better culture medium than KSOM for efficient in vitro production (IVP of bovine embryos.

  1. Effect of supplementation of different growth factors in embryo culture medium with a small number of bovine embryos on in vitro embryo development and quality.

    Science.gov (United States)

    Ahumada, C J; Salvador, I; Cebrian-Serrano, A; Lopera, R; Silvestre, M A

    2013-03-01

    When embryos are cultured individually or in small groups, blastocyst yield efficiency and quality are usually reduced. The aim of this work was to investigate the effect of supplementation of the embryo culture medium (CM) with several growth factors (GFs) on embryo development and apoptosis rate when a reduced number of embryos were in vitro cultured. Two experimental studies (ES) were carried out. In ES 1, five treatments were tested to study the effect of GF on embryo development: Control (∼30 to 50 embryos cultured in 500 μl of CM); Control 5 (Five embryos cultured in 50 μl microdrops of CM), without addition of GF in either of the two control groups; epidermal GF (EGF); IGF-I; and transforming GF-α (TGF-α) (Five embryos were cultured in 50 μl microdrops of CM with 10 ng/ml EGF, 10 ng/ml IGF-I or 10 ng/ml TGF-α, respectively). In ES 2, following the results obtained in ES 1, four different treatments were tested to study their effect on embryo development and quality (number of cells per blastocyst and apoptotic rate): Control; Control 5; EGF, all three similar to ES 1; EGF + IGF-I group (five embryos cultured in 50 μl microdrops of CM with 10 ng/ml EGF and 10 ng/ml IGF-I). In both ESs, it was observed that a higher proportion of embryos cultured in larger groups achieved blastocyst stage than embryos cultured in reduced groups (22.6% v. 14.0%, 12.6% and 5.3% for Control v. Control 5, IGF-I, TGF-α groups in ES 1, and 24.9% v. 17.1% and 19.0% for Control v. Control 5 and EGF in ES 2, respectively; P embryos cultured in medium supplemented with EGF (18.5%) or with EGF + IGF-I (23.5%), in ES 1 and ES 2, respectively. With regard to blastocyst quality, embryos cultured in reduced groups and supplemented with EGF, alone or combined with IGF-I, presented lower apoptosis rates than embryos cultured in reduced groups without GF supplementation (11.6% and 10.5% v. 21.9% for EGF, EGF + IGF-I and Control 5 groups, respectively; P embryos, increasing blastocyst

  2. Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

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

  3. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  4. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael

    2006-01-01

    on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  5. Bystander effect in human hepatoma HepG2 cells caused by medium transfers at different times after high-LET carbon ion irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Wu Qingfeng [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate School of Chinese Academy of Sciences, Beijing 100039 (China); Li Qiang, E-mail: liqiang@impcas.ac.c [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Jin Xiaodong; Liu Xinguo [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Dai Zhongying [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Graduate School of Chinese Academy of Sciences, Beijing 100039 (China)

    2011-01-15

    Although radiation-induced bystander effects have been well documented in a variety of biological systems, whether irradiated cells have the ability to generate bystander signaling persistently is still unclear and the clinical relevance of bystander effects in radiotherapy remains to be elucidated. This study examines tumor cellular bystander response to autologous medium from cell culture irradiated with high-linear energy transfer (LET) heavy ions at a therapeutically relevant dose in terms of clonogenic cell survival. In vitro experiments were performed using human hepatoma HepG2 cell line exposed to 100 keV/{mu}m carbon ions at a dose of 2 Gy. Two different periods (2 and 12 h) after irradiation, irradiated cell conditioned medium (ICCM) and replenished fresh medium were harvested and then transferred to unirradiated bystander cells. Cellular bystander responses were measured with the different medium transfer protocols. Significant higher survival fractions of unirradiated cells receiving the media from the irradiated cultures at the different times post-irradiation than those of the control were observed. Even replenishing fresh medium for unirradiated cells which had been exposed to the ICCM for 12 h could not prevent the bystander cells from the increased survival fraction. These results suggest that the irradiated cells could release unidentified signal factor(s), which induced the increase in survival fraction for the unirradiated bystander cells, into the media sustainedly and the carbon ions triggered a cascade of signaling events in the irradiated cells rather than secreting the soluble signal factor(s) just at a short period after irradiation. Based on the observations in this study, the importance of bystander effect in clinical radiotherapy was discussed and incorporating the bystander effect into the current radiobiological models, which are applicable to heavy ion radiotherapy, is needed urgently.

  6. Bystander effect in human hepatoma HepG2 cells caused by medium transfers at different times after high-LET carbon ion irradiation

    International Nuclear Information System (INIS)

    Wu Qingfeng; Li Qiang; Jin Xiaodong; Liu Xinguo; Dai Zhongying

    2011-01-01

    Although radiation-induced bystander effects have been well documented in a variety of biological systems, whether irradiated cells have the ability to generate bystander signaling persistently is still unclear and the clinical relevance of bystander effects in radiotherapy remains to be elucidated. This study examines tumor cellular bystander response to autologous medium from cell culture irradiated with high-linear energy transfer (LET) heavy ions at a therapeutically relevant dose in terms of clonogenic cell survival. In vitro experiments were performed using human hepatoma HepG2 cell line exposed to 100 keV/μm carbon ions at a dose of 2 Gy. Two different periods (2 and 12 h) after irradiation, irradiated cell conditioned medium (ICCM) and replenished fresh medium were harvested and then transferred to unirradiated bystander cells. Cellular bystander responses were measured with the different medium transfer protocols. Significant higher survival fractions of unirradiated cells receiving the media from the irradiated cultures at the different times post-irradiation than those of the control were observed. Even replenishing fresh medium for unirradiated cells which had been exposed to the ICCM for 12 h could not prevent the bystander cells from the increased survival fraction. These results suggest that the irradiated cells could release unidentified signal factor(s), which induced the increase in survival fraction for the unirradiated bystander cells, into the media sustainedly and the carbon ions triggered a cascade of signaling events in the irradiated cells rather than secreting the soluble signal factor(s) just at a short period after irradiation. Based on the observations in this study, the importance of bystander effect in clinical radiotherapy was discussed and incorporating the bystander effect into the current radiobiological models, which are applicable to heavy ion radiotherapy, is needed urgently.

  7. Bovine serum albumin contained in culture medium used in artificial insemination is an important anaphylaxis risk factor.

    Science.gov (United States)

    Pagán, Juan A; Postigo, Idoia; Rodríguez-Pacheco, Jorge R; Peña, Maribel; Guisantes, Jorge A; Martínez, Jorge

    2008-11-01

    To analyze the cause of the anaphylactic reaction after a standard artificial insemination process in a patient diagnosed with asthma. Case report. Residencia Sanitaria Virgen de la Arrixaca (Murcia, Spain) and University of the Basque Country (Vitoria, Spain). A 30-year-old woman with a previous medical history compatible with respiratory allergy who suffered an anaphylactic reaction after an artificial insemination with spermatozoids in capable medium (Upgraded B2 INRA medium; Laboratories CCD, Paris, France). Cutaneous tests and specific IgE levels to inhalant allergens, grass and Olea pollens, and insemination medium were performed. Specific IgE levels to mammal epithelia and bovine serum albumin (BSA). Skin prick tests were positive for inhalant allergens such as mites, cat, dog, horse, and rabbit epithelia, grasses and Olea pollens, and the insemination medium. The beta-lactamic tests were negative. The determination of specific IgE demonstrated positive values to mammal epithelia and mammal serum albumins including BSA. We report a case of an anaphylactic reaction to the BSA included in the insemination culture medium induced by a subclinical sensitivity to serum albumins of mammal epithelia. A previous testing with the medium is recommended and specific testing might be needed in women who have a history of animal epithelium allergies.

  8. Chemical properties and antioxidant activity of exopolysaccharides fractions from mycelial culture of Inonotus obliquus in a ground corn stover medium.

    Science.gov (United States)

    Xiang, Yuling; Xu, Xiangqun; Li, Juan

    2012-10-15

    The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. We first reported the enhancement in production and antioxidant activity of exopolysaccharides by I. obliquus culture under lignocellulose decomposition. In this study, the two different sources of exopolysaccharides from the control medium and the lignocellulose (corn stover) containing medium by I. obliquus in submerged fermentation were fractionated and purified by chromatography. The exopolysaccharides from the corn stover-containing medium presented significantly stronger hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity than the control. Three fractions from the control medium and the corn stover-containing medium were isolated respectively. The fraction of DEPL3 from the corn stover-containing medium with the highest protein content (38.3%), mannose content (49.6%), and the lowest molecular weight (29 kDa) had the highest antioxidant activity with the lowest IC50 values. In conclusion, lignocellulose decomposition changed the chemical characterisation and significantly enhanced the antioxidant activity of exopolysaccharide fractions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Effect of water potential on sclerotial production by Sclerotinia sclerotiorum in a culture medium

    Science.gov (United States)

    Sclerotinia sclerotiorum causes Sclerotinia blight on peanut. Potato dextrose agar medium was prepared and adjusted to various water potentials (-0.4 to -3.4 MPa) using NaCl. Petri plates (9-cm dia) each containing 15 ml of medium were inoculated with a 4-mm agar plug of S. sclerotiorum. Plates w...

  10. Rat bone marrow stem cells isolation and culture as a bone formative experimental system

    Directory of Open Access Journals (Sweden)

    Amer Smajilagić

    2013-02-01

    Full Text Available Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with multipotential potential and differentiation in to the different cells types such as are osteoblast, chondroblast, adipoblast. In this research we describe pioneering experiment of tissue engineering in Bosnia and Herzegovina, of the isolation and differentiation rat bone marrow stromal cells in to the osteoblast cells lineages. Rat bone marrow stromal cells were isolated by method described by Maniatopulos using their plastic adherence capatibility. The cells obtained by plastic adherence were cultured and serially passaged in the osteoinductive medium to differentiate into the osteocytes. Bone marrow samples from rats long bones used for isolation of stromal cells (BMSCs. Under determinate culture conditions BMSCs were differentiated in osteogenic cell lines detected by Alizarin red staining three weeks after isolation. BMSCs as autologue cells model showed high osteogenetic potential and calcification capatibility in vitro. In future should be used as alternative method for bone transplantation in Regenerative Medicine.

  11. Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

    Science.gov (United States)

    Cao, Ting-Ting; Zhang, Yu-Qing

    2015-09-01

    Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

  12. Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

    Directory of Open Access Journals (Sweden)

    Haiying Guo

    2018-01-01

    Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

  13. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization

    DEFF Research Database (Denmark)

    Ziebe, Søren; Loft, Anne; Povlsen, Betina B

    2013-01-01

    To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR).......To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR)....

  14. Culture environment regulates amino acid turnover and glucose utilisation in human ES cells.

    Science.gov (United States)

    Rathjen, Joy; Yeo, Christine; Yap, Charlotte; Tan, Boon Siang Nicholas; Rathjen, Peter D; Gardner, David K

    2014-06-01

    Human embryonic stem (ES) cells have been proposed as a renewable source of pluripotent cells that can be differentiated into various cell types for use in research, drug discovery and in the emerging area of regenerative medicine. Exploitation of this potential will require the development of ES cell culture conditions that promote pluripotency and a normal cell metabolism, and quality control parameters that measure these outcomes. There is, however, relatively little known about the metabolism of pluripotent cells or the impact of culture environment and differentiation on their metabolic pathways. The effect of two commonly used medium supplements and cell differentiation on metabolic indicators in human ES cells were examined. Medium modifications and differentiation were compared in a chemically defined and feeder-independent culture system. Adding serum increased glucose utilisation and altered amino acid turnover by the cells, as well as inducing a small proportion of the cells to differentiate. Cell differentiation could be mitigated by inhibiting p38 mitogen-activated protein kinase (p38 MAPK activity). The addition of Knockout Serum Replacer also incr