Continuous Cell Culture Research Articles

Rice gall dwarf virus exploits tubules to facilitate viral spread among cultured insect vector cells derived from leafhopper Recilia dorsalis

Rice gall dwarf virus (RGDV), a member of the family Reoviridae, causes repeated epidemics in rice fields in southern China. An RGDV isolate collected from Guangdong Province (southern China) is mainly transmitted by leafhopper vector Recilia dorsalis in a persistent-propagative manner. The infection by RGDV induces the formation of virus-containing tubules in the plant host and insect vector. In this study, we established continuous cell cultures of the leafhopper R. dorsalis to investigate the functional role of these tubules within the insect vector. Cytopathologic studies revealed that the tubules, which comprised viral non-structural protein Pns11 and contained viral particles, were able to protrude from the surface of cultured leafhopper cells. Tubule-like structures formed in non-host insect cells after the expression of Pns11 in a baculovirus system, suggesting that Pns11 was the minimal viral factor required for the formation of the tubules induced by RGDV infection. In cultured leafhopper cells, knockdown of Pns11 expression from RNA interference, induced by synthesized dsRNA from the Pns11 gene, abolished the formation of such tubules, preventing the direct cell-to-cell spread of RGDV without significant effects on viral multiplication. All these results show that RGDV exploits virus-containing tubules to facilitate viral spread among its insect vector cells.

Accelerating animal cell growth in perfusion mode by multivariable control: simulation studies

This study considers the problem of manipulating in an optimal way the perfusion and bleed flow rates of a continuous culture of hybridoma cells, so as to achieve a fast transient start-up and reject potential disturbances. The proposed solution makes use of an analysis of the properties of the steady state solutions of the nonlinear dynamic model of the cell culture, and in particular the relationship between the two main limiting substrates, glucose and glutamine. The solution is implemented using extended prediction self-adaptive control. Simulation results demonstrate the approach potentiality.

A high-throughput electrochemical impedance spectroscopy evaluation of bioresponsibility of the titanium microelectrode array integrated with hydroxyapatite and silver

This paper reports a transparent Ti microelectrode array (MEA) system for a high-throughput evaluation of bioresponsibility using electrochemical impedance spectroscopy (EIS). The MEA chip integrated with hydroxyapatite (HA) and Ag coatings was selectively prepared by electrochemical deposition based on a novel procedure of multichannel current control. The EIS measurement of living MG63 osteosarcoma cells in the integrated MEA chip was conducted, and the result was analyzed using an equivalent circuit corresponding to a titanium oxide film, protein adsorption layer, cell adhesion layer, and medium. It is shown that the bioresponsibility of Ti–Ag–HA on the MEA chip can be improved, compared with the Ti, Ti–HA, and Ti–Ag coatings. The system was further used for real-time EIS monitoring during continuous cell culture for a long period (12 days). The effect of the long-term cell proliferation on the EIS behavior was discussed. This integrated system is valuable to significantly simplify the operation procedures and quickly evaluate the bioresponsibility of biomaterials.

Fed‐batch and perfusion culture processes: Economic, environmental, and operational feasibility under uncertainty

This article evaluates the current and future potential of batch and continuous cell culture technologies via a case study based on the commercial manufacture of monoclonal antibodies. The case study compares fed-batch culture to two perfusion technologies: spin-filter perfusion and an emerging perfusion technology utilizing alternating tangential flow (ATF) perfusion. The operational, economic, and environmental feasibility of whole bioprocesses based on these systems was evaluated using a prototype dynamic decision-support tool built at UCL encompassing process economics, discrete-event simulation and uncertainty analysis, and combined with a multi-attribute decision-making technique so as to enable a holistic assessment. The strategies were compared across a range of scales and titres so as to visualize how their ranking changes in different industry scenarios. The deterministic analysis indicated that the ATF perfusion strategy has the potential to offer cost of goods savings of 20% when compared to conventional fed-batch manufacturing processes when a fivefold increase in maximum viable cell densities was assumed. Savings were also seen when the ATF cell density dropped to a threefold increase over the fed-batch strategy for most combinations of titres and production scales. In contrast, the fed-batch strategy performed better in terms of environmental sustainability with a lower water and consumable usage profile. The impact of uncertainty and failure rates on the feasibility of the strategies was explored using Monte Carlo simulation. The risk analysis results demonstrated the enhanced robustness of the fed-batch process but also highlighted that the ATF process was still the most cost-effective option even under uncertainty. The multi-attribute decision-making analysis provided insight into the limited use of spin-filter perfusion strategies in industry. The resulting sensitivity spider plots enabled identification of the critical ratio of weightings of economic and operational benefits that affect the choice between ATF perfusion and fed-batch strategies.

Human immunodeficiency virus enhances hepatitis C virus replication by differential regulation of IFN and TGF family genes

HIV co-infection significantly impacts the natural history of hepatitis C virus (HCV) by increasing plasma HCV viral load, accelerating liver disease progression, and reducing rates of HCV clearance. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the impact of HIV on cytokine expression is unknown. In this study, an HCV continuous infection cell culture system was modified to permit co-infection with HIV to test the hypothesis that virus-induced disregulation of immune-response genes, particularly interferons and TGF-β, may create a permissive environment for the initial establishment of HIV/HCV co-infection in the host. CCR5-expressing Huh-7.5 hepatoma cells were transduced with human CD4 antigen to allow HIV infection in vitro. Co-infection of CD4⁺ Huh-7.5 cells with HIV and HCV or co-culture of HIV-infected CD4⁺ Huh-7.5 cells and HCV-infected Huh-7.5 cells increased the level of HCV RNA compared to HCV mono-infection. Quantitative gene expression analysis revealed HIV-induced up regulation of most tested IFN family genes when compared to HCV or co-infection. HCV infection induced up regulation of many TGF family genes that were subsequently down-regulated in the presence of HIV or HIV/HCV. Interestingly, co-infection resulted in down regulation of several IFN genes and significant up regulation of TGF-β genes leading to an overall enhancement of HCV replication. These data suggest that HIV infection may influence HCV replication in vitro by increasing levels of HCV RNA, possibly through the differential regulation of endogenous IFN and TGF family genes.

Parallelized microfluidic biochips in multi well plate applied to liver tissue engineering

Abstract In this paper, we present a tool box to perform parallelized microfluidic cultures. The box was made in polycarbonate (PC) using a 24-well plate format. The microfluidic biochips were made in polydimethylsiloxane (PDMS). Two wells of the PC plate were used as inlet/outlet and fluid reservoir for one biochip. A hermetic cover was fabricated in PC to allow continuous and sterile dynamic cell cultures. Thus, the entire set up, called IDCCM for “Integrated Dynamic Cell Cultures in Microsystems” allowed the perfusion culture of 12 biochips simultaneously. To demonstrate the potential of the IDCCM box and the cell functionality, we analyzed the behaviour of HepG2/C3a liver cells. For that purpose, we have investigated the effects of the inoculated cell density and of the flow rates on the cell proliferation, glucose consumption, albumin production and the cytochrome P450-1A activity over 96 h of cultures. The results have shown that the cell proliferation and the cytochrome P450-1A activity were cell density and flow rate dependent. This led to identify a best optimized condition with HepG2/C3a at (5–7) × 105 cells/biochip (corresponding to (2.5–3.5) × 105 cells/cm2) and at a perfusion flow rate of 25 μL/min. This result highlighted the functionality of the HepG2/C3a cells in parallelized microfluidic culture conditions and illustrated the potential for larger in vitro toxicity studies using the IDCCM tools.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

A new large‐scale manufacturing platform for complex biopharmaceuticals

Complex biopharmaceuticals, such as recombinant blood coagulation factors, are addressing critical medical needs and represent a growing multibillion-dollar market. For commercial manufacturing of such, sometimes inherently unstable, molecules it is important to minimize product residence time in non-ideal milieu in order to obtain acceptable yields and consistently high product quality. Continuous perfusion cell culture allows minimization of residence time in the bioreactor, but also brings unique challenges in product recovery, which requires innovative solutions. In order to maximize yield, process efficiency, facility and equipment utilization, we have developed, scaled-up and successfully implemented a new integrated manufacturing platform in commercial scale. This platform consists of a (semi-)continuous cell separation process based on a disposable flow path and integrated with the upstream perfusion operation, followed by membrane chromatography on large-scale adsorber capsules in rapid cycling mode. Implementation of the platform at commercial scale for a new product candidate led to a yield improvement of 40% compared to the conventional process technology, while product quality has been shown to be more consistently high. Over 1,000,000 L of cell culture harvest have been processed with 100% success rate to date, demonstrating the robustness of the new platform process in GMP manufacturing. While membrane chromatography is well established for polishing in flow-through mode, this is its first commercial-scale application for bind/elute chromatography in the biopharmaceutical industry and demonstrates its potential in particular for manufacturing of potent, low-dose biopharmaceuticals.

Establishment of human ovarian serous carcinomas cell lines in serum free media

Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts [19], Ricci-Vitiani et al. [20], Cammareri et al. [21]).Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors that recreate the morphology of the original patient tumor when implanted in immune deficient mice. The introduction of this method will facilitate the expansion of ovarian cancer cells for investigating cancer stem cell biology as well as providing tools to aid in the development of new treatments for this deadly disease.

Different NF-κB activation characteristics of human respiratory syncytial virus subgroups A and B

Human respiratory syncytial virus (HRSV) is a member of the family Paramyxoviridae, and is responsible for serious respiratory illness in infants, the elderly and the immunocompromised. HRSV exists as two distinct lineages known as subgroups A and B, which represent two lines of divergent evolution with extensive genetic and serologic differences. While both subgroup A and B viruses contribute to overall HRSV disease, subgroup A isolates are associated with both increased frequency and morbidity of infections, and reasons for this are unclear. HRSV disease is characterized by virus-mediated cell destruction in combination with extensive inflammatory and immune modulatory responses, and for HRSV subgroup A isolates, several of these signaling pathways are regulated through activation of the transcription factor NF-κB. In contrast, the NF-κB activation characteristics of HRSV subgroup B infection remain untested. Here, we performed a quantitative and comparative analysis of NF-κB activation in response to infection of both continuous and primary cell cultures with HRSV subgroup A and B isolates. Our results showed the model HRSV subgroup A isolate consistently induced increased NF-κB activation compared to its HRSV subgroup B counterpart. The differential NF-κB activation characteristics of HRSV subgroup A and B viruses may contribute to differences in their pathogenesis.

Development of disposable PDMS micro cell culture analog devices with photopolymerizable hydrogel encapsulating living cells

Microscale cell culture devices with two or more cell types, such as the micro cell culture analog (microCCA), are promising devices to predict mammalian response to toxic drug and chemical exposure. A polydimethylsiloxane (PDMS) version of such microfluidic devices has been challenging to construct due to the difficulty of patterning multi cell types directly into designated individual cell culture chambers in an oxygen plasma bonded PDMS device. Approaches with micro-valves for flow control are complex, expensive and inconvenient to use. In this study, an alternative approach using polyethylene glycol diacrylate (PEG-DA) for spatially controlled multi-cell type patterning inside a bonded microCCA device is described. We constructed a three-cell type PDMS microCCA following a human physiologically based pharmacokinetic (PBPK) modeling, and applied continuous cell culture medium recirculation within the device as a blood surrogate. A fluorescence microscope based direct pattern writing method was used to form cell/hydrogel microstructures with higher cell viability than the traditional UV lamp based method. The positive effect of mixed molecular weight PDG-DA on hydrogel-encapsulated cell membrane integrity was also studied. This prototype PDMS microCCA device was then tested with Triton X-100 as a model toxicant. The combination of hydrogel photo-patterning and the microfluidic cell culture platform enables the fabrication of simple and low cost multi-cell type biosensors for drug development, toxicity study and clinical diagnosis.

Characterization of the Interaction between Human Respiratory Syncytial Virus and the Cell Cycle in Continuous Cell Culture and Primary Human Airway Epithelial Cells

Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.

Increased consistency and efficiency in routine potency testing by bioassay with direct use of cryopreserved (ready-to-plate) cells

The use of cells as a cryopreserved, readily available reagent has facilitated high-throughput screening of new drug candidates by bioassay. This practice is considerably less labor intensive and allows more flexibility in laboratory testing than traditional continuous cell culture. We have shown that this practice can be adapted to cell proliferation and reporter gene assay formats used in routine sample testing for determination of relative potency of commercial product in a Quality Control Laboratory. The ability to use the same, optimized population of cells provides consistency in an assay over time. Measures of assay performance to indicate maintenance of the validated state of a method and to determine benefit on variation in potency results were compared between cultured and cryopreserved (frozen ready-to-plate) cells. Control of the cellular component, which is the most variable aspect of most cell based potency assays, allowed detection of more minor contributors to variability. In a cell proliferation assay format, the final result was a highly precise bioassay (repeatability CV of 2%). An improvement in process capability (Cp) was noted when ready-to-plate cells were used in the studies completed over long periods of time.

The molecular action of the novel insecticide, Pyridalyl

Pyridalyl is a recently discovered insecticide that exhibits high insecticidal activity against Lepidoptera and Thysanoptera. Pyridalyl action requires cytochrome P450 activity, possibly for production of a bioactive derivative, Pyridalyl metabolism being prevented by general P450 inhibitors. Apoptosis is apparently not involved in the cytotoxicity. Continuous culture of Spodoptera frugiperda Sf21 cells in sub-lethal doses of Pyridalyl, results in a Pyridalyl-resistant cell line. Probing the molecular action of Pyridalyl by comparison of the proteomes of Pyridalyl-resistant and –susceptible cell lines, revealed differential expression of a number of proteins, including the up-regulation of thiol peroxiredoxin (TPx), in the resistant cells. Treatment of Bombyx mori larvae with Pyridalyl, followed by comparison of the midgut microsomal sub-proteome, revealed the up-regulation of three proteasome subunits. Such subunits, together with Hsp70 stress proteins, glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) and thiol peroxiredoxin (TPx) were also up-regulated in the whole proteome of B. mori BM36 cells following treatment with the insecticide. The foregoing results lead to the hypothesis that cytochrome P450 action leads to an active Pyridalyl metabolite, which results in production of reactive oxygen species (ROS), that leads to damage to cellular macromolecules (e.g., proteins) and enhanced proteasome activity leads to increased protein degradation and necrotic cell death.

Migratory capabilities of human umbilical cord blood-derived neural stem cells (HUCB-NSC) in vitro.

Many types of neural progenitors from various sources have been evaluated for therapy of CNS disorders. Prerequisite for success in cell therapy is the ability for transplanted cells to reach appropriate target such as stroke lesion. We have established neural stem cell line from human umbilical cord blood neural stem (HUCB-NSC). In the present study we evaluated migratory capabilities of cells (HUCB-NSC) and the presence of various migration-related receptors. Immunocytochemical analysis revealed abundant expression of CXCR4, PDGFR-alpha, PDGFR-beta, c-Met, VEGFR, IGF-1R and PSA-NCAM receptors in non-adherent population of HUCB-NSC cultured in serum free (SF) conditions (SF cells). Biological activity of selected receptors was confirmed by HUCB-NSC in vitro migration towards SDF-1 and IGF-1 ligands. Additionally, rat brain-derived homogenates have been assessed for their chemoattractive activity of HUCB-NSC. Our experiments unveiled that brain tissue was more attracted for HUCB-NSC than single ligands with higher potency of injured than intact brain. Moreover, adherent HUCB-NSC cultured in low serum (LS) conditions (LS cells) were employed to investigate an impact of different extracellular matrix (ECM) proteins on cell motility. It turned out that laminin provided most permissive microenvironment for cell migration, followed by fibronectin and gelatin. Unexpected nuclear localization of CXCR4 in SF cells prompted us to characterize intracellular pattern of this expression in relation to developmental stage of cells cultured in different conditions. Continuous culture of LS cells revealed cytoplasmatic pattern of CXCR4 expression while HUCB-NSC cultured in high serum conditions (HS cells) resulted in gradual translocation of CXCR4 from nucleus to cytoplasm and then to arising processes. Terminal differentiation of HUCB-NSC was followed by CXCR4 expression decline.

Continuous CHO cell cultures with improved recombinant protein productivity by using mannose as carbon source: Metabolic analysis and scale-up simulation

The replacement of glucose by mannose as a means to improve recombinant protein productivity was studied for the first time in continuous cultures of Chinese hamster ovary (CHO) cells producing human recombinant tissue plasminogen activator (t-PA). Steady-state operation at two hexose levels in the inlet (2.5 and 10 mM) allowed comparing the effect of sugar type and concentration on cell metabolism and t-PA production independently of changes in specific growth rates produced by different culture conditions. An increase in biomass concentration (15–20%) was observed when using mannose instead of glucose. Moreover, specific hexose consumption rates were 20–25% lower in mannose cultures whereas specific production rates of lactate, an undesirable by-product, were 25–35% lower than in glucose control cultures. The volumetric productivity of t-PA increased up to 30% in 10 mM mannose cultures, without affecting the sialylation levels of the protein. This increase is manly explained by the higher cell concentration, and represents a substantial improvement in the t-PA production process using glucose. Under this condition, the oxygen uptake rate and the specific oxygen consumption rate, both estimated by a stoichiometric analysis, were about 10% and 25% lower in mannose cultures, respectively. These differences lead to significant differences at larger scales, as it was estimated by simulating cell cultures at different bioreactor sizes (5–5000 L). By assuming a set of regular operating conditions in this kind of process, it was determined that mannose-based cultures could allow culturing CHO cells up to 3000 L compared to only 80 L in glucose cultures at the same conditions. These facts indicate that mannose cultures may have a significant advantage over glucose cultures not only in terms of volumetric productivity of the recombinant protein but also for their potential application in large-scale productive processes.

Toxoplasma gondii RH Ankara: Production of evolving tachyzoites using a novel cell culture method

Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem.The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5×106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3×106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12×106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55×106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31–0.37×106/ml, respectively.ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains.

Yeast‐mediated stereo‐selective reduction of estrone by continuous cell culture with dual stirred tanks for product yield improvement

AbstractBACKGROUND: β‐Estradiol is an important hormone for the treatment of breast cancer and osteoporosis. β‐Estradiol can be produced via Saccharomyces cerevisiae mediated reduction of estrone. However, substrate inhibition and low production yield have been observed in the batch cell culture.RESULTS: An innovative continuous cell culture with dual stirred tanks in series was designed to solve the above problems. The growth medium was fed continuously to the incubation tank where the cells were incubated aerobically; the viable cells were then supplied continuously to the reaction tank in which the yeast‐mediated anaerobic reduction of estrone was performed with continuous feed of the substrate medium and continuous withdawal of the reaction cell culture. Thus, an increase in cell productivity from about 3‐fold to 7‐fold was obtained when compared with the batch cell culture. The β‐estradiol yield was improved to 64.8% on the second reaction day, accompanied by an accumulation of 12.9 mg β‐estradiol on the third reaction day. The yield was about 10% more and the accumulated recovery of β‐estradiol was 4.3‐fold better than with the batch cell culture. The diastereomeric excess value (%de) of β‐estradiol was more than 99%.CONCLUSION: A high product yield with excellent stereo‐selectivity was achieved in a short reaction period with the developed continuous cell culture and the dual stirred tank. Copyright © 2010 Society of Chemical Industry

Condition of Mild Hypothermia does not Promote an Increase in Specific Productivity of Recombinant Protein in Continuous Culture of CHO Cells

Condition of Mild Hypothermia does not Promote an Increase in Specific Productivity of Recombinant Protein in Continuous Culture of CHO Cells

P136. Effects of mycoplasma elimination agent on cardiac differentiation of human embryonic stem cells

Mycoplasma contamination is a major problem in continuous cell culture. Various agents are used for the elimination of mycoplasma. However, their effects on cardiac differentiation potential of human embryonic stem cells (hESCs) are rarely known. In this study, we tried to evaluate the effects of mycoplasma elimination agent (plasmocin) on differentiation into cardiomyocytes (CMs) from hESCs. Undifferentiated hESCs were induced to differentiated into CMs in the presence of P/S and plasmocin (2.5, 5.0, 10.0 μg/ml). The expression of cardiac specific genes such as Nkx2.5, Tbx5, α-myosin heavy chain (MHC) and cardiac troponin (cTn) I was evaluated using immunocytochemistry and qPCR. After 14 days of differentiation, the beating cluster formation of hESCs was reduced in plasmocin-treated group. The expression of cardiac genes was down-regulated and the decrease of cTn I was significant in plasmocin-treated group. In this study, we demonstrated the effects of mycoplasma elimination agent on cardiac differentiation potential of hESCs. There results showed that addition of mycoplasma elimination agent reduced the differentiation potential of hESCs (Nos. 2010-0016819 and SC1150).