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Sample records for serum-free cho cell

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

    PubMed

    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.

  2. Adaptation of CHO cells in serum-free conditions for erythropoietin production: Application of EVOP technique for process optimization.

    PubMed

    Jukić, Suzana; Bubenik, Dijana; Pavlović, Nediljko; Tušek, Ana Jurinjak; Srček, Višnja Gaurina

    2016-09-01

    Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum- or protein-free conditions early in their development. This is a labor- and time-consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum- and protein-free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein-free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein-free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  3. Stable production of a human growth hormone antagonist from CHO cells adapted to serum-free suspension culture.

    PubMed

    Haldankar, R; Kopchick, J J; Ridgway, D

    1999-01-01

    Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kDa. An hGH analogue was created with a single amino acid substitution (glycine[G] 120 to arginine[R]) in the third alpha-helix of the hGH molecule. This hGH analogue, named hGHG120R, was found to be an hGH antagonist. It is a parenteral drug candidate for treating conditions in which hGH levels are abnormally high, as found in type I diabetics. Previously, a genetically engineered anchorage-dependent mouse L cell line was created that produced and secreted hGHG120R in culture media (Dulbecco's modified Eagle's medium, DMEM) supplemented with 5% NuSerum IV. A multistep downstream process was developed to purify hGHG120R. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, size exclusion chromatography, reversed phase high-performance liquid chromatography, phase separation, and lyophilization. Here, we present the development of a superior eukaryotic system using a proper combination of genetic elements, cell line, and media formulation. This system is suitable for the large-scale production of the recombinant protein and is superior to the previously developed system in that it increases the specific production rate and at the same time eases the burden of the purification process, in both time and efficiency. Dihydrofolate reductase mutant (DHFR-) Chinese hamster ovary (CHO) cells were used that were stably transfected with an expression vector in which the hGHG120R gene is driven by the relatively strong human cytomegalovirus-early gene regulatory region. The hGHG120R tested to be biologically active. These cells were then adapted to grow in suspension in CHO-S-SFM (serum-free media). High cell densities, typically 2.0 x 10(6) cells/mL were obtained from spinner flask cultures. Partial purification of hGHG120R from CHO cell cultured media revealed that the level of impurities in SFM was significantly lower than the serum

  4. Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments.

    PubMed

    Kim, Sung Hyun; Lee, Gyun Min

    2009-06-01

    An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L(-1)) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert(R). The resulting medium gave a 1.9-3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.

  5. Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19.

    PubMed

    Park, Ju Hyun; Wang, Zesong; Jeong, Hee-Jin; Park, Hee Ho; Kim, Byung-Gee; Tan, Wen-Song; Choi, Shin Sik; Park, Tai Hyun

    2012-11-01

    We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of

  6. Novel Cell-Ess ® supplement used as a feed or as an initial boost to CHO serum free media results in a significant increase in protein yield and production.

    PubMed

    Elhofy, Adam

    2016-01-01

    Many metrics, including metabolic profiles, have been used to analyze cell health and optimize productivity. In this study, we investigated the ability of a lipid supplement to increase protein yield. At a concentration of 1% (v/v) the lipid supplement caused a significant increase in protein titer (1118 ± 65.4 ng 10(5) cells(- 1) days(- 1)) when compared to cultures grown in the absence of supplementation (819.3 ± 38.1 ng 10(5) cells(- 1) days(- 1); p < 0.05). This equated to a 37% increase in productivity. Furthermore, metabolic profiles of ammonia, glutamate, lactate, and glucose were not significantly altered by the polar lipid supplement. In a separate set of experiments, using the supplement as a feed resulted in 2 notable effects. The first was a 25% increase in protein titer. The second was an extension of peak protein production from 1 day to 2 days. These results suggest that lipid supplementation is a promising avenue for enhancing protein production. In addition, our results also suggest that an increase in protein production may not necessarily require a change in the metabolic state of the cells.

  7. Serum-free solutions for cryopreservation of cells.

    PubMed

    Campbell, Lia H; Brockbank, Kelvin G M

    2007-01-01

    With the development of cell-based assays and therapies, the purity of reagents used to grow and maintain cells has become much more important. In particular, the use of fetal calf serum for culturing cells presents a direct path for potential contamination of cell cultures. In recent years, much research has focused on the development of serum-free culturing systems, not only to alleviate difficulties due to availability and cost of fetal calf serum but also to prevent the transmission of potentially fatal diseases to human patients. Additionally, methods need to be developed for long-term storage of cell stocks that also reduce the risk of exposure to harmful diseases. As most methods employ fetal calf serum in their freezing formulations, solutions that avoid the use of fetal calf serum while providing equivalent or better recovery of cells upon thawing would be ideal. In this study, two vascular cell lines have been cryopreserved as adherent cell populations in two widely used cryoprotectants, dimethyl sulfoxide and 1,2-propanediol, and two vehicle solutions, Euro-Collins and Unisol-cryoprotectant vehicle specifically formulated for the maintenance of cell homeostasis at temperatures below 37 degrees C. The addition of serum to these formulations was also evaluated to determine if its presence provided any additional benefit to the cells during cryopreservation. The results demonstrated that using vehicle solutions designed for lower temperatures produced viable cells that retained cell population viability values up to 75% of unfrozen controls. These results also demonstrated that including serum in the formulation provided no additional benefit to the cells and in some cases actually produced lower cell viability after cryopreservation. In conclusion, the development of solutions designed for low-temperature storage of cells provides a viable alternative to more conventional cryopreservation protocols and eliminates the necessity of including serum in these

  8. Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.

    PubMed

    Ogawa, Akiko; Mizui, Shinya; Chida, Yasuhito; Shimizu, Masafumi; Terada, Satoshi; Ohura, Takeshi; Kobayashi, Kyo-Ichi; Yasukawa, Saori; Moriyama, Nobuyuki

    2014-07-01

    Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Short communication: pitfalls of culturing C8166 cells in serum-free media.

    PubMed

    Teo, Ian; Choi, Ji-Won; Buttigieg, Karen; Shaunak, Sunil

    2005-03-01

    Long-term culture of C8166 cells in serum-free media can result in changes in their level of expression of immunologically important cell surface makers and a loss of infectivity by HIV-1. We have now demonstrated that these phenotypic changes are due to an outgrowth of a very small number of contaminating cells of mouse origin. Our observations emphasize the importance of carefully recharacterizing any cells that have been adapted to grow in a serum-free culture media.

  10. SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture.

    PubMed

    Schlaeger, Ernst-Jürgen; Kitas, Eric A; Dorn, Arnulf

    2003-05-01

    A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 mug/ml) were found 3-4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS-PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents.

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

    PubMed

    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.

  12. Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines.

    PubMed

    Huang, Ding; Peng, Wen-Juan; Ye, Qian; Liu, Xu-Ping; Zhao, Liang; Fan, Li; Xia-Hou, Kang; Jia, Han-Jing; Luo, Jian; Zhou, Lin-Ting; Li, Bei-Bei; Wang, Shi-Lei; Xu, Wen-Ting; Chen, Ze; Tan, Wen-Song

    2015-01-01

    Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted.

  13. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    USDA-ARS?s Scientific Manuscript database

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  14. CHO glycosylation mutants as potential host cells to produce therapeutic proteins with enhanced efficacy.

    PubMed

    Zhang, Peiqing; Chan, Kah Fai; Haryadi, Ryan; Bardor, Muriel; Song, Zhiwei

    2013-01-01

    CHO glycosylation mutants, pioneered by Stanley and co-workers, have proven to be valuable tools in glycobiology and biopharmaceutical research. Here we aim to provide a summary of our efforts to isolate industrially applicable CHO glycosylation mutants, termed CHO-gmt cells, using cytotoxic lectins and zinc-finger nuclease technology. The genetic defects in the glycosylation machinery in these cells lead to the production of recombinant glycoproteins with consistent and unique glycan structures. In addition, these mutant cells can be easily adapted to serum-free medium in suspension cultures, the condition used by the biotech industry for large-scale production of recombinant therapeutics. In light of the critical impact of glycosylation on biopharmaceutical performances, namely, safety and efficacy, the CHO-gmt lines have enormous potential in producing glycoprotein therapeutics with optimal glycosylation profiles, thus, representing a panel of ideal host cell lines for producing recombinant biopharmaceuticals with improved safety profiles and enhanced efficacy.

  15. Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium.

    PubMed

    da Costa-Silva, Thaís Alves; da Silva Meira, Cristina; Frazzatti-Gallina, Neuza; Pereira-Chioccola, Vera Lucia

    2012-04-01

    Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different

  16. A serum-free medium developed for in vitro expansion of murine intestinal stem cells.

    PubMed

    Mohamed, Mahmoud S; Chen, Yun; Yao, Chao-Ling

    2014-07-01

    Intestinal stem cells (ISCs) are located at the base of the intestinal crypts and have the ability to self-renew as well as to differentiate into mature epithelial cells. Recently, ISCs have received much attention for the treatment of many intestinal diseases. However, many challenges face those studying ISCs because insufficient ISCs are available. Therefore, the development of a culture medium for ISC expansion is an important necessity for basic research and clinical application. In this study, we described the technique used to develop a serum-free medium for expanding ISCs in vitro. Furthermore, five serum substitutes were selected and optimized in order to maintain the long-term proliferation and enteroid-forming ability of ISCs: (i) ethanolamine; (ii) ascorbic acid phosphate; (iii) transferrin; (iv) glutathione; and (v) sodium selenite. Analysis of gene expression of Lgr5, Bmi1, Msi1 and PTEN demonstrated that our serum-free medium sustained the expression of genes involved in ISC-related functions in the expanded ISCs. Additionally, the expression intensity of surface markers, including Lgr5, CD24 and CD44, on serum-free expanded cells in crypts was greatly increased. Taken together, our results demonstrate that the number of ISCs can be expanded and their functionality maintained in our serum-free medium, indicating the suitability of this serum-free expansion medium for increasing the numbers of ICSs available for basic research and clinical applications in the future. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. [Adherent and single-cell suspension culture of Madin-Darby canine kidney cells in serum-free medium].

    PubMed

    Huang, Ding; Zhao, Liang; Tan, Wensong

    2011-04-01

    In recent years, there are tremendous economic and social losses across the world because of virus-related diseases. It is well known that Madin-Darby canine kidney (MDCK) cells are easily handled, quickly amplified and efficiently infected with influenza virus. Therefore, they are considered as one of the most important cell lines for the production of influenza vaccine. In this work, we first developed a serum-free adherent culture process for MDCK cells with an in-house prepared serum-free medium MDCK-SFM. Next, we derived a cell line named ssf-MDCK, which was amenable for single-cell suspension culture in the serum-free medium. We found that during serum-free batch culture of MDCK cells, the peak viable cell density and maximum specific growth rate were 3.81 x 10(6) cells/mL and 0.056 h(-1), respectively; 3.6- and 1.6-fold increase compared with those in serum-containing adherent batch culture. In addition, we compared growth and metabolic characteristics of MDCK cells in serum-containing adherent culture, serum-free adherent culture and serum-free single-cell suspension culture. We found that less metabolic by-products were produced in both serum-free cultures. In serum-free single-cell suspension batch culture, the viable cell density was highest. These results are critical for establishing large-scale suspension culture of MDCK cells as subsequent well as large-scale influenza vaccine production.

  18. Chemically defined serum-free conditions for cartilage regeneration from human embryonic stem cells.

    PubMed

    Yang, Dandan; Chen, Shubin; Gao, Changzhao; Liu, Xiaobo; Zhou, Yulai; Liu, Pengfei; Cai, Jinglei

    2016-11-01

    The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF β3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Production of influenza virus in serum-free mammalian cell cultures.

    PubMed

    Merten, O W; Manuguerra, J C; Hannoun, C; van der Werf, S

    1999-01-01

    Human influenza viruses are routinely isolated and grown in a variety of mammalian cell substrates. However, influenza viruses for use as inactivated vaccine are still produced in embryonated eggs. Using a perfusion culture-based bioreactor process using serum-free medium, both human and equine influenza viruses of different types and subtypes could be produced to high titres. Classical DEAE-dextran microcarriers were found to be more suitable than polyester sponge carriers for virus production. In addition, MDCK cells grown in serum-free medium were further validated as the most suitable cell substrate compared to Vero and BHK-21 C13 cells for large scale virus production of influenza virus. Finally, to minimize potential contamination by adventitious agents, it was demonstrated that a new serum-free medium in which all animal-derived products are replaced by a plant extract, efficiently supports the growth of MDCK cells as well as the production of influenza virus in the presence of trypsin when using the perfusion bioreactor process.

  20. Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells

    PubMed Central

    Tüysüz, Nesrin; van Bloois, Louis; van den Brink, Stieneke; Begthel, Harry; Verstegen, Monique M. A.; Cruz, Luis J.; Hui, Lijian; van der Laan, Luc J. W.; de Jonge, Jeroen; Vries, Robert; Braakman, Eric; Mastrobattista, Enrico; Cornelissen, Jan J.; Clevers, Hans; ten Berge, Derk

    2017-01-01

    Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein. PMID:28262686

  1. Kinetic characterization of vero cell metabolism in a serum-free batch culture process.

    PubMed

    Petiot, Emma; Guedon, Emmanuel; Blanchard, Fabrice; Gény, Cécile; Pinton, Hervé; Marc, Annie

    2010-09-01

    A global kinetic study of the central metabolism of Vero cells cultivated in a serum-free medium is proposed in the present work. Central metabolism including glycolysis, glutaminolysis, and tricarboxylic acid cycle (TCA) was demonstrated to be saturated by high flow rates of consumption of the two major substrates, glucose, and glutamine. Saturation was reavealed by an accumulation of metabolic intermediates and amino acids, by a high production of lactate needed to balance the redox pathway, and by a low participation of the carbon flow to the TCA cycle supply. Different culture conditions were set up to reduce the central metabolism saturation and to better balance the metabolic flow rates between lactate production and energetic pathways. From these culture conditions, substitutions of glutamine by other carbon sources, which have lower transport rates such as asparagine, or pyruvate in order to shunt the glycolysis pathway, were successful to better balance the central metabolism. As a result, an increase of the cell growth with a concomitant decrease of cell death and a better distribution of the carbon flow between TCA cycle and lactate production occurred. We also demonstrated that glutamine was a major carbon source to supply the TCA cycle in Vero cells and that a reduction of lactate production did not necessary improve the efficiency of the Vero cell metabolism. Thus, to adapt the formulation of the medium to the Vero cell needs, it is important to provide carbon substrates inducing a regulated supply of carbon in the TCA cycle either through the glycolysis or through other pathways such as glutaminolysis. Finally, this study allowed to better understand the Vero cell behavior in serum-free medium which is a valuable help for the implementation of this cell line in serum-free industrial production processes.

  2. Synthetic promoters for CHO cell engineering.

    PubMed

    Brown, Adam J; Sweeney, Bernie; Mainwaring, David O; James, David C

    2014-08-01

    We describe for the first time the creation of a library of 140 synthetic promoters specifically designed to regulate the expression of recombinant genes in CHO cells. Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks. The most active second generation promoters achieved approximately twice the activity of a control construct harboring the human cytomegalovirus (CMV) promoter. Lastly, we evaluated the function of a subset of synthetic promoters exhibiting a broad range of activity in

  3. [Evaluation of the critical process parameters for the cultivation of recombinant Chinese hamster ovary cells in serum-free fed-batch mode].

    PubMed

    Liu, Xingmao; Liu, Hong; Ye, Lingling; Li, Shichong; Wu, Benchuan; Wang, Qiwei; Chen, Zhaolie

    2011-02-01

    Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.

  4. Survival and neurite growth of chick embryo spinal cord cells in serum-free culture.

    PubMed

    Tanaka, H; Obata, K

    1982-07-01

    Cell survival and neurite growth were investigated in serum-free spinal cord cell cultures on polyornithine coating (PORN). Cells were obtained from 6- or 7-day-old chick embryos. Isolated spinal cord cells required promoting factors for their survival and neurite growth. The survival-promoting factors were initially present in spinal cord cells. High density cultures, co-cultures with spinal cord explants, and spinal cord extract promoted survival of isolated spinal cord cells in MEM with no additives. Other tissue extracts (brain, liver, heart and skeletal muscle), serum, and serum-free conditioned medium (SF-CM) of muscle or glioma C6 cells also promoted survival. The active substances in the brain extract and SF-CM were shown to be protein and were separated into 3 fractions (approximately molecular weight 150,000, 70,000, 40,000) by gel filtration chromatography. Survival and neurite growth were suggested to be promoted by different factors because: (1) survival was promoted by both tissue extract and SF-CM, but neurite growth was promoted only by SF-CM; (2) the neurite growth-stimulating activity of SF-CM was lost following dialysis and heat (100 degrees C, 2 min) treatment; however, the survival-promoting activity was not. It was also suggested that spinal cord cells produce neurite growth promoting factors, but did not initially contain these factors.

  5. Misonidazole-glutathione conjugates in CHO cells

    SciTech Connect

    Varghese, A.J.; Whitmore, G.F.

    1984-08-01

    Misonidazole, after reduction to the hydroxylamine derivative, reacts with glutathione (GSH) under physiological conditions. The reaction product has been identified as a mixture of two isomeric conjugates. When water soluble extracts of CHO cells exposed to misonidazole under hypoxic conditions are subjected to HPLC analysis, misonidazole derivatives, having the same chromatographic properties as the GSH-MISO conjugates, were detected. When CHO cells were incubated with misonidazole in the presence of added GSH, a substantial increase in the amount of the conjugate was detected. When extracts of CHO cells exposed to misonidazole under hypoxia were subsequently exposed to GSH, an increased formation of the conjugate was observed. A rearrangement product of the hydroxylamine derivative of misonidazole is postulated as the reactive intermediate responsible for the formation of the conjugate.

  6. A signature of 12 microRNAs is robustly associated with growth rate in a variety of CHO cell lines.

    PubMed

    Klanert, Gerald; Jadhav, Vaibhav; Shanmukam, Vinoth; Diendorfer, Andreas; Karbiener, Michael; Scheideler, Marcel; Bort, Juan Hernández; Grillari, Johannes; Hackl, Matthias; Borth, Nicole

    2016-10-10

    As Chinese Hamster Ovary (CHO) cells are the cell line of choice for the production of human-like recombinant proteins, there is interest in genetic optimization of host cell lines to overcome certain limitations in their growth rate and protein secretion. At the same time, a detailed understanding of these processes could be used to advantage by identification of marker transcripts that characterize states of performance. In this context, microRNAs (miRNAs) that exhibit a robust correlation to the growth rate of CHO cells were determined by analyzing miRNA expression profiles in a comprehensive collection of 46 samples including CHO-K1, CHO-S and CHO-DUKXB11, which were adapted to various culture conditions, and analyzed in different growth stages using microarrays. By applying Spearman or Pearson correlation coefficient criteria of>|0.6|, miRNAs with high correlation to the overall growth, or growth rates observed in exponential, serum-free, and serum-free exponential phase were identified. An overlap of twelve miRNAs common for all sample sets was revealed, with nine positively and three negatively correlating miRNAs. The here identified panel of miRNAs can help to understand growth regulation in CHO cells and contains putative engineering targets as well as biomarkers for cell lines with advantageous growth characteristics.

  7. Development of Serum-Free Media for Lepidopteran Insect Cell Lines.

    PubMed

    Chan, Leslie C L; Reid, Steven

    2016-01-01

    Baculovirus-based Insect Cell Technology (ICT) is widely used for the expression of recombinant heterologous proteins and baculovirus bioinsecticides, and has recently gained momentum as a commercial manufacturing platform for human and veterinary vaccines. The three key components of ICT are the Lepidopteran insect cell line, the baculovirus vector, and the growth medium. Insect cell growth media have evolved significantly in the past five decades, from basal media supplemented with hemolymph or animal serum, to highly optimized serum-free media and feeds (SFM and SFF) capable of supporting very high cell densities and recombinant protein yields. The substitution of animal sera with protein hydrolysates in SFM results in greatly reduced medium costs and much improved process scalability. However, both sera and hydrolysates share the disadvantage of lot-to-lot variability, which is detrimental to process reproducibility. Hence, the industrialization of ICT would benefit greatly from chemically defined media (CDM) for insect cells, which are not yet commercially available. On the other hand, applications such as baculovirus bioinsecticides would need truly low cost serum-free media and feeds (LC-SFM and LC-SFF) for economic viability, which require the substitution of a majority of expensive added amino acids with even higher levels of hydrolysates, hence increasing the risk of a variable process. CDM developments are anticipated to benefit both conventional and low cost ICT applications, by identifying key growth factors in hydrolysates for more targeted media and feed design.

  8. Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

    SciTech Connect

    Deslex, S.; Negrel, R.; Ailhaud, G.

    1987-01-01

    Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IFG-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, (/sup 14/)glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and and GPDH. The development of a serum free, chemically defined medium for the differentiation of diploid adiopose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.

  9. Heparin promotes suspension adaptation process of CHO-TS28 cells by eliminating cell aggregation.

    PubMed

    Li, Ling; Qin, Jun; Feng, Qiang; Tang, Hao; Liu, Rong; Xu, Liqing; Chen, Zhinan

    2011-01-01

    While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO-TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10(4) cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO-TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.

  10. Serum-reduced and serum-free media for differentiation of Caco-2 cells.

    PubMed

    Ferruzza, Simonetta; Rossi, Carlotta; Sambuy, Yula; Scarino, Maria Laura

    2013-01-01

    Human intestinal Caco-2 cells were differentiated using serum-reduced medium with fetal bovine serum (FBS) added only to the basolateral (BL) medium, and four serum-free media, containing insulin, transferrin, selenium (ITS), or MITO+™ serum extender (ITS plus growth factors), with or without addition of a lipid mixture, respectively. Differentiation was assessed by monitoring monolayer permeability, alkaline phosphatase and sucrase activities, and the transport of digoxin and cephalexin. Notably, the serum-reduced protocol produced results that were comparable to cells differentiated in the control medium and should be recommended as an alternative to the use of 10% FBS in both apical (AP) and BL media. ITS serum-free medium elicited permeability values and cephalexin transport similar to control cells. MITO+™ medium was the most efficient in promoting the two transport activities investigated, and it should be further evaluated with a larger set of substances, although its undisclosed composition represents a limit that may override these advantages.

  11. Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture.

    PubMed

    Minamoto, Y; Ogawa, K; Abe, H; Iochi, Y; Mitsugi, K

    1991-01-01

    We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium.As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37°C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640+10% FBS.It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable.Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast.Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2×10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day.The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.

  12. Development of a serum-free and heat-sterilizable medium and continuous high-density cell culture.

    PubMed

    Minamoto, Y; Ogawa, K; Abe, H; Iochi, Y; Mitsugi, K

    1991-01-01

    We tried to establish a new serum-free and heat-sterilizable medium, based on our serum-free medium in which many lymphoblastoid cells and hybridoma could grow as well as in a conventional serum-containing medium. As is well-known, L-glutamine (L-Gln) is one of the most heat-labile but essential components for cell growth. As a substitute for L-Gln, dipeptide such as Gly-L-Gln or L-Ala-L-Gln, which was quite stable even after autoclaving, was found to be utilizable for mammalian cell growth. The L-Gln dipeptide-containing serum-free medium was quite stable in a solution even after storing at 37 degrees C for 4 months. In the serum-free medium containing L-Ala-L-Gln, mouse hybridola could grow and produce more antibody than in RPMI 1640 + 10% FBS. It has been proved that BSA and transferrin, which are also heat-labile but essential for the growth of various cell lines, can be substituted by heat-stable alpha-cyclodextrin and cholesterol, and Fe-gluconate, respectively. Insulin has also proved to be heat stable in a solution of Fe-gluconate. We thus established a new serum-free medium, all the components of which could be heat-sterilizable. Moreover, by adding EGF and BSA but without the adhesion factor included in FBS, the serum-free medium was found to support a long-term serial culture of a human diploid fibroblast. Finally, with this auotoclavable serum-free medium in a perfusion culture apparatus, we were able to continuously cultivate a human lymphoblastoid cell line. The production rate of IgM was found to be markedly increased by feeding the serum-free medium enriched by glucose, bicarbonate, L-Cys, and approtinin. The cell density reached as high as 2 x 10(8)/ml in the serum-free medium. Although the working volume in the reactor was only 1 1, the rate of IgM production reached 480 mg/day. The new heat-sterilizable serum-free medium has several advantages, because L-Gln peptide is a heat-stable and available precursor of L-Gln.

  13. Xenobiotic- and Serum-Free Culture of Oral Mucosal Epithelial Cells on Contact Lenses.

    PubMed

    Björkblom, Benny; Eidet, Jon R; Utheim, Tor P; Ulltveit-Moe, Harald F; Raeder, Sten

    2016-01-01

    Cultured autologous oral mucosal epithelial cells (OMECs) have proven useful in the treatment of ocular surface disorders. This study is the first to investigate the potential of expanding OMEC in a xenobiotic- and serum-free medium using therapeutic contact lenses (CLs) as a substrate and carrier. Porcine OMEC were seeded on laminin-coated lotrafilcon A therapeutic CLs with the density of 8 × 10(4) cells/lens and cultured in a defined serum and xenobiotic-free medium. Confocal immunofluorescence microscopy was used to analyze the following: (1) cellular morphology by using rhodamine-phalloidin staining of F-actin, (2) phenotype by applying antibodies against the progenitor cell marker p63 and the putative stem cell marker ABCG2 and (3) cell viability by using propidium iodide and Hoechst 33342 dual staining. Porcine OMEC attached well to the CLs, and cell-to-cell contacts were evident. After three days in culture, the OMEC displayed a confluent monolayer with uniform cobblestone morphology, whereas stratified cultures with 2-3 layers were formed after six days. No significant difference in expression of p63 was observed after three-day culture (79.4 ± 14.8%) compared with six-day culture (60.3 ± 18.9%). ABCG2 expression in the basal cell layer was 6.3 ± 1.0% and 4.8 ± 1.8% after three- and six-day culture, respectively. The basal layer viability of cultured OMECs was 99.3 ± 0.2% and 82.8 ± 1.1% after three and six days culture, respectively. The use of therapeutic CLs has potential as a substrate and carrier for OMEC cultured in a xenobiotic- and serum-free culture system.

  14. Separation of CHO cells using hydrocyclones

    PubMed Central

    Pinto, Rodrigo C.V.; Castilho, Leda R.

    2007-01-01

    Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs. PMID:19002842

  15. Separation of CHO cells using hydrocyclones.

    PubMed

    Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

    2008-01-01

    Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

  16. Serum-free media for the production of human mesenchymal stromal cells: a review.

    PubMed

    Gottipamula, S; Muttigi, M S; Kolkundkar, U; Seetharam, R N

    2013-12-01

    The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.

  17. Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

    PubMed Central

    Jung, Sunghoon; Panchalingam, Krishna M.; Rosenberg, Lawrence; Behie, Leo A.

    2012-01-01

    Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies. PMID:22645619

  18. Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process.

    PubMed

    Heathman, Thomas R J; Glyn, Veronica A M; Picken, Andrew; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

    2015-08-01

    Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation. © 2015 The Authors. Biotechnology and Bioengineering

  19. Gene expression changes during Giardia-host cell interactions in serum-free medium.

    PubMed

    Ferella, Marcela; Davids, Barbara J; Cipriano, Michael J; Birkeland, Shanda R; Palm, Daniel; Gillin, Frances D; McArthur, Andrew G; Svärd, Staffan

    2014-10-01

    Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.

  20. Death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

    PubMed Central

    1991-01-01

    Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, are proastroblasts that are acutely dependent on epidermal growth factor (EGF) for survival. Ultrastructurally, an early change found in SFME cells deprived of EGF was a loss of polysomes which sedimentation analysis confirmed to be a shift from polysomes to monosomes. The ribosomal shift was not accompanied by decreased steady-state level of cytoplasmic actin mRNA examined as an indicator of cellular mRNA level. With time the cells became small and severely degenerate and exhibited nuclear morphology characteristic of apoptosis. Genomic DNA isolated from cultures undergoing EGF deprivation-dependent cell death exhibited a pattern of fragmentation resulting from endonuclease activation characteristic of cells undergoing apoptosis or programmed cell death. Flow cytometric analysis indicated that cultures in the absence of EGF contained almost exclusively G1-phase cells. Some of the phenomena associated with EGF deprivation of SFME cells are similar to those observed upon NGF deprivation of nerve cells in culture, suggesting that these neuroectodermal-derived cell types share common mechanisms of proliferative control involving peptide growth factor-dependent survival. PMID:2016341

  1. Evaluation of Serum-Free, Xeno-Free Cryopreservation Solutions for Human Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Miyagi-Shiohira, Chika; Kobayashi, Naoya; Saitoh, Issei; Watanabe, Masami; Noguchi, Yasufumi; Matsushita, Masayuki; Noguchi, Hirofumi

    2017-01-01

    Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine. PMID:28174671

  2. Characterisation of novel uveal melanoma cell lines under serum-free conditions.

    PubMed

    Suesskind, D; Gauss, S; Faust, U E A; Bauer, P; Schrader, M; Bartz-Schmidt, K U; Henke-Fahle, S

    2013-08-01

    The establishment of long-term uveal melanoma (UM) cell lines is difficult. However, studying living cells and their behaviour in the presence of other cells and the extracellular matrix is important in terms of understanding tumour biology and malignant behaviour. We have established three UM cell lines and report a first characterisation of these cell lines. Three established UM cell lines (UMT2, UMT26 and UMT33) were analysed according to their morphologic characteristics, melanocytic differentiation, adhesion on different extracellular matrices and proliferative activity. Copy number changes of chromosomes 1, 3, 6 and 8 were studied by multiplex ligation-dependent probe amplification (MLPA). Oncogenic mutations in UM involving exons 4 and 5 of GNAQ and GNA11, respectively, were analysed by sequencing. All cell lines grew in suspension. UMT2 cells were homogeneous, UMT26 and UMT33 cells heterogeneous with regard to cell size and pigmentation. All UM cell lines revealed a melanocytic differentiation. UMT2 and 33 adhered on various extracellular matrices, while UMT26 only adhered to basal membrane extract (BME). This difference corresponded to the different expression of various integrins. Ki67 was expressed by 89% of UMT2 and 95% of UMT33 cells, which thus were in a proliferative stage, while only 2% of UMT26 cells revealed immunostaining for this proliferation marker. The doubling time of UMT2 was 3 days, 12 days for UMT33, and circa 3-4 months for UMT26. MLPA revealed disomy 3 in UMT2 and monosomy 3 in UMT33. The same point mutation was found in UMT2, 26 and 33, in exon 5 of GNA11 at codon 209 (p.Q209L). The establishment of UM cell lines under serum-free conditions is possible. Characterisation of UMT2, 26, and 33 revealed obvious differences in cytomorphology, melanocytic differentiation, adhesion on extracellular matrices, and proliferative activity. UMT2, 26 and 33 showed the same oncogenic mutation in exon 5 of GNA11.

  3. Human adipose derived stroma/stem cells grow in serum-free medium as floating spheres.

    PubMed

    Dromard, C; Bourin, P; André, M; De Barros, S; Casteilla, L; Planat-Benard, V

    2011-04-01

    With the goal of obtaining clinically safe human adipose-derived stroma/stem cells (ASC) and eliminating the use of serum, we have developed a new culture system that allows the expansion of ASC as spheres in a defined medium. These spheres can be passaged several times. They are not only aggregated cells but rather originate from single cells as clonal spheres can be obtained after seeding at very low density and reform clonal spheres after dissociation. These spheres can also revert to monolayer growth when plated in medium containing human plasma and even generate fibroblast-like colonies (CFU-f). Under several differentiation-specific media, spheres-derived ASC maintain their capacity to differentiate into osteoblasts, endothelial cells and adipocytes. These results indicate that human ASC can be maintained in a serum-free 3D culture system, which is of great interest for the expansion in bioreactors of autologous ASC and their use in clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    PubMed

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

  5. A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

    NASA Technical Reports Server (NTRS)

    Chuman, L. M.; FINE; COHEN; Saier, M. H.

    1985-01-01

    The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

  6. A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

    NASA Technical Reports Server (NTRS)

    Chuman, L. M.; FINE; COHEN; Saier, M. H.

    1985-01-01

    The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

  7. The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells.

    PubMed

    Costa, Ana Rita; Withers, Joanne; Rodrigues, Maria Elisa; McLoughlin, Niaobh; Henriques, Mariana; Oliveira, Rosário; Rudd, Pauline M; Azeredo, Joana

    2013-06-25

    N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

  8. Performance of serum-supplemented and serum-free media in IFNgamma Elispot Assays for human T cells.

    PubMed

    Janetzki, Sylvia; Price, L; Britten, C M; van der Burg, S H; Caterini, J; Currier, J R; Ferrari, G; Gouttefangeas, C; Hayes, P; Kaempgen, E; Lennerz, V; Nihlmark, K; Souza, V; Hoos, A

    2010-04-01

    The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories' own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNgamma-secretion.

  9. A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures.

    PubMed

    Mohamed, S N; Holmes, R; Hartzell, C R

    1983-06-01

    A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.

  10. Cell engineering and cultivation of chinese hamster ovary (CHO) cells.

    PubMed

    Omasa, Takeshi; Onitsuka, Masayoshi; Kim, Wook-Dong

    2010-04-01

    Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

  11. Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells

    PubMed Central

    Davami, Fatemeh; Baldi, Lucia; Rajendra, Yashas; M. Wurm, Florian

    2014-01-01

    The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells. PMID:25317401

  12. Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells

    PubMed Central

    Sefcik, Lauren S.; Neal, Rebekah A.; Kaszuba, Stephanie N.; Parker, Anna M.; Katz, Adam J.; Ogle, Roy C.; Botchwey, Edward A.

    2011-01-01

    Electrospinning has recently gained widespread attention as a process capable of producing nanoscale fibres that mimic native extracellular matrix. In this study, we compared the osteogenic differentiation behaviour of human adipose stem cells (ASCs) on a 3D nanofibre matrix of type I rat tail collagen (RTC) and a 2D RTC collagen-coated substrate, using a novel serum-free osteogenic medium. The serum-free medium significantly enhanced the numbers of proliferating cells in culture, compared to ASCs in traditional basal medium containing 10% animal serum, highlighting a potential clinical role for in vitro stem cell expansion. Osteogenic differentiation behaviour was assessed at days 7, 14 and 21 using quantitative real-time RT–PCR analysis of the osteogenic genes collagen I (Coll I), alkaline phosphatase (ALP), osteopontin (OP), osteonectin (ON), osteocalcin (OC) and core-binding factor-α (cbfa1). All genes were upregulated (>one-fold) in ASCs cultured on nanofibre scaffolds over 2D collagen coatings by day 21. Synthesis of mineralized extracellular matrix on the scaffolds was assessed on day 21 with Alizarin red staining. These studies demonstrate that 3D nanoscale morphology plays a critical role in regulating cell fate processes and in vitro osteogenic differentiation of ASCs under serum-free conditions. PMID:18493910

  13. [Spheres isolated from Colo205 cell line possess cancer stem-like cells under serum-free culture condition].

    PubMed

    Li, Ying-fei; Xiao, Bing; Lai, Zhuo-sheng; Tu, San-fang; Wang, Yuan-yuan; Zhang, Xiao-lan

    2008-02-01

    Isolation and expansion tumor spheres from colorectal cancer cell line Colo205 cultured in serum-free medium(SFM) supplemented with human recombinant EGF and bFGF. Colo205 cells were cultivated in SFM,while cells cultivated in serum-supplemented medium(SSM) served as the control. Cells morphology were observed by optical microscope, and expression of intestinal stem cells marker Musashi-1 was detected by immunocytochemical. To induce cell differentiation, tumour spheres were cultivated without EGF and bFGF in the presence of 10% serum. Then we analysed expressions of stem cell surface markers CD133 and CD44 among undifferentiated cell, post-differentiated cells and routine Colo205 cells under serum-supplemented culture condition by flow cytometry. At last we compared cell cycle and spectral karyotype between two groups. In SFM consisting of EGF and bFGF, a minority of Colo205 cells could survive, proliferate and form the suspended tumor spheres. We detected high Musashi-1 expression in these cells. Compared with the SSM group and the post-differentiation SFM group, the expressions of CD133 and CD44 were significantly increased in the undifferentiated SFM group (P<0.05). There was no statistical difference in the expression of CD133 and CD44 between the post-differentiation SFM group and the SSM group (P>0.05). Cell cycle analysis indicated that tumor spheres were of a high proliferation state.We could not find any noticeable difference in the number of chromatosomes between the SFM group and the SSM group. Tumor spheres in which enriched cancer stem cells can be generated under serum-free culture condition with EGF and bFGF.

  14. Development of serum-free quality and quantity control culture of colony-forming endothelial progenitor cell for vasculogenesis.

    PubMed

    Masuda, Haruchika; Iwasaki, Hiroto; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Ishikawa, Masakazu; Ii, Masaaki; Shizuno, Tomoko; Sato, Atsuko; Ito, Rie; Horii, Miki; Ishida, Hideyuki; Kato, Shunichi; Asahara, Takayuki

    2012-02-01

    Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133(+) cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration.

  15. Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

    PubMed Central

    Masuda, Haruchika; Iwasaki, Hiroto; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Ishikawa, Masakazu; Ii, Masaaki; Shizuno, Tomoko; Sato, Atsuko; Ito, Rie; Horii, Miki; Ishida, Hideyuki; Kato, Shunichi

    2012-01-01

    Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture (QQc) in terms of the most vasculogenic colony-forming EPC expansion, evaluated by the newly established EPC colony formation assay. The QQc of umbilical cord blood-CD133+ cells for 7 days produced a 52.9-fold increase in total cell number and 3.28-fold frequency in definitive EPC colony development, resulting in a 203.9-fold increase in estimated total definitive EPC colony number in vitro. Pre- or post-QQc cells were intramyocardially transplanted into nude rats with myocardial infarction (MI). Echocardiographic and micromanometer-tipped conductance catheter examinations 28 days post-MI revealed significant preservation of left ventricular (LV) function in rats receiving pre- or post-QQc cells compared with those receiving phosphate-buffered saline. Assessments of global LV contractility indicated a dose-dependent effect of pre- or post-QQc cells and the superior potency of post-QQc cells over pre-QQc cells. Furthermore, immunohistochemistry showed more abundant formation of both human and rat endothelial cells and cardiomyocytes in the infarcted myocardium following transplantation of post-QQc cells compared with pre-QQc cells. Our optimal serum-free quality and quantity culture may enhance the therapeutic potential of EPCs in both quantitative and qualitative aspects for cardiovascular regeneration. PMID:23197763

  16. Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine.

    PubMed

    Murabayashi, Dai; Mochizuki, Mai; Tamaki, Yuichi; Nakahara, Taka

    2017-07-01

    Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

  17. Toward stable gene expression in CHO cells

    PubMed Central

    Mariati; Koh, Esther YC; Yeo, Jessna HM; Ho, Steven CL; Yang, Yuansheng

    2014-01-01

    Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific. PMID:25482237

  18. Glycosaminoglycans affect heparanase location in CHO cell lines.

    PubMed

    Piva, Maria B R; Suarez, Eloah R; Melo, Carina M; Cavalheiro, Renan P; Nader, Helena B; Pinhal, Maria A S

    2015-09-01

    Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

  19. Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Muttigi, Manjunatha S; Chaansa, S; Ashwin, K M; Priya, Nancy; Kolkundkar, Udaykumar; SundarRaj, Swathi; Majumdar, Anish Sen; Seetharam, Raviraja N

    2016-02-01

    The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

  20. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    PubMed

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  1. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Ashwin, K M; Muttigi, Manjunatha S; Kannan, Suresh; Kolkundkar, Udaykumar; Seetharam, Raviraja N

    2014-04-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

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

    PubMed

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

    2017-09-19

    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 < 0.05), with lower percentages of CD90(+) cells found in equine MSC samples 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

  3. Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture.

    PubMed

    Aihara, Yuko; Hayashi, Yohei; Hirata, Mitsuhi; Ariki, Nobutaka; Shibata, Shinsuke; Nagoshi, Narihito; Nakanishi, Mio; Ohnuma, Kiyoshi; Warashina, Masaki; Michiue, Tatsuo; Uchiyama, Hideho; Okano, Hideyuki; Asashima, Makoto; Furue, Miho Kusuda

    2010-01-01

    The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

  4. Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

    PubMed Central

    2011-01-01

    Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

  5. Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures.

    PubMed

    Kim, Hun; Lee, Su Jeen; Park, Jin Yong; Park, Yong Wook; Kim, Hyun Sung; Kang, Heui-Yun; Hur, Byung-Ki; Ryu, Yeon-Woo; Han, Sang In; Kim, Jong Su

    2004-03-01

    Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine

  6. Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium.

    PubMed

    Zainal Ariffin, Shahrul Hisham; Kermani, Shabnam; Zainol Abidin, Intan Zarina; Megat Abdul Wahab, Rohaya; Yamamoto, Zulham; Senafi, Sahidan; Zainal Ariffin, Zaidah; Abdul Razak, Mohamad

    2013-01-01

    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.

  7. Dynamic transcription programs during ES cell differentiation towards mesoderm in serum versus serum-freeBMP4 culture.

    PubMed

    Bruce, Stephen J; Gardiner, Brooke B; Burke, Les J; Gongora, M Milena; Grimmond, Sean M; Perkins, Andrew C

    2007-10-10

    Expression profiling of embryonic stem (ES) cell differentiation in the presence of serum has been performed previously. It remains unclear if transcriptional activation is dependent on complex growth factor mixtures in serum or whether this process is intrinsic to ES cells once the stem cell program has been inactivated. The aims of this study were to determine the transcriptional programs associated with the stem cell state and to characterize mesoderm differentiation between serum and serum-free culture. ES cells were differentiated as embryoid bodies in 10% FBS or serum-free media containing BMP4 (2 ng/ml), and expression profiled using 47 K Illumina(R) Sentrix arrays. Statistical methods were employed to define gene sets characteristic of stem cell, epiblast and primitive streak programs. Although the initial differentiation profile was similar between the two culture conditions, cardiac gene expression was inhibited in serum whereas blood gene expression was enhanced. Also, expression of many members of the Kruppel-like factor (KLF) family of transcription factors changed dramatically during the first few days of differentiation. KLF2 and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, dynamic changes in KLF-DNA binding activities occurred upon differentiation, and strong bio-informatic evidence for direct regulation of many stem cell genes by KLFs was found. Down regulation of stem cell genes and activation of epiblast/primitive streak genes is similar in serum and defined media, but subsequent mesoderm differentiation is strongly influenced by the composition of the media. In addition, KLF family members are likely to be important regulators of many stem cell genes.

  8. Dynamic transcription programs during ES cell differentiation towards mesoderm in serum versus serum-freeBMP4 culture

    PubMed Central

    Bruce, Stephen J; Gardiner, Brooke B; Burke, Les J; Gongora, M Milena; Grimmond, Sean M; Perkins, Andrew C

    2007-01-01

    Background Expression profiling of embryonic stem (ES) cell differentiation in the presence of serum has been performed previously. It remains unclear if transcriptional activation is dependent on complex growth factor mixtures in serum or whether this process is intrinsic to ES cells once the stem cell program has been inactivated. The aims of this study were to determine the transcriptional programs associated with the stem cell state and to characterize mesoderm differentiation between serum and serum-free culture. Results ES cells were differentiated as embryoid bodies in 10% FBS or serum-free media containing BMP4 (2 ng/ml), and expression profiled using 47 K Illumina(R) Sentrix arrays. Statistical methods were employed to define gene sets characteristic of stem cell, epiblast and primitive streak programs. Although the initial differentiation profile was similar between the two culture conditions, cardiac gene expression was inhibited in serum whereas blood gene expression was enhanced. Also, expression of many members of the Kruppel-like factor (KLF) family of transcription factors changed dramatically during the first few days of differentiation. KLF2 and KLF4 co-localized with OCT4 in a sub-nuclear compartment of ES cells, dynamic changes in KLF-DNA binding activities occurred upon differentiation, and strong bio-informatic evidence for direct regulation of many stem cell genes by KLFs was found. Conclusion Down regulation of stem cell genes and activation of epiblast/primitive streak genes is similar in serum and defined media, but subsequent mesoderm differentiation is strongly influenced by the composition of the media. In addition, KLF family members are likely to be important regulators of many stem cell genes. PMID:17925037

  9. Shift of microRNA profile upon glioma cell migration using patient-derived spheroids and serum-free conditions.

    PubMed

    Munthe, Sune; Halle, Bo; Boldt, Henning B; Christiansen, Helle; Schmidt, Steffen; Kaimal, Vivek; Xu, Jessica; Zabludoff, Sonya; Mollenhauer, Jan; Poulsen, Frantz R; Kristensen, Bjarne W

    2017-03-01

    Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The miRNA profiling revealed 30 miRNAs to be differentially expressed. In total 13 miRNAs were upregulated and 17 downregulated in migrating cells compared to corresponding spheroids. The three most deregulated miRNAs, miR-1227 (up-regulated), miR-32 (down-regulated) and miR-222 (down-regulated), were experimentally overexpressed. A non-significantly increased migration rate was observed after miR-1227 overexpression. A significantly reduced migration rate was observed after miR-32 and miR-222 overexpression. In conclusion a shift in microRNA profile upon glioma cell migration was identified using an assay avoiding serum-induced migration. Both the miRNA profiling and the functional validation suggested that miR-1227 may be associated with increased migration and miR-32 and miR-222 with decreased migration. These miRNAs may represent potential novel targets in migrating glioma cells.

  10. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

    PubMed

    Broussau, Sophie; Jabbour, Nadine; Lachapelle, Guillaume; Durocher, Yves; Tom, Rosanne; Transfiguracion, Julia; Gilbert, Rénald; Massie, Bernard

    2008-03-01

    We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

  11. Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics.

    PubMed

    Greiner, J F W; Hauser, S; Widera, D; Müller, J; Qunneis, F; Zander, C; Martin, I; Mallah, J; Schuetzmann, D; Prante, C; Schwarze, H; Prohaska, W; Beyer, A; Rott, K; Hütten, A; Gölzhäuser, A; Sudhoff, H; Kaltschmidt, C; Kaltschmidt, B

    2011-12-17

    Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

  12. Benefits and Limitations of Protein Hydrolysates as Components of Serum-Free Media for Animal Cell Culture Applications

    NASA Astrophysics Data System (ADS)

    Lobo-Alfonso, Juliet; Price, Paul; Jayme, David

    Increased understanding of influential factors for the cultivation of animal cells, combined with heightened regulatory concern over potential transmission of adventitious contaminants associated with serum and other animal-derived components, has elevated interest in using protein hydrolysates as serum replacements or nutrient supplements. This paper reviews the chemistry and biology of various hydrolysates derived from animal, plant and microbial sources. It provides specific examples of a beneficial selection of plant and yeast hydrolysates as ingredients of serum-free nutrient formulations for bioproduction applications of cultured mammalian and insect cells. Strategies for customizing and optimizing nutrients for specialized applications and general benefits and limitations of protein hydrolysates for biopharmaceutical production are also discussed.

  13. Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells

    PubMed Central

    Ramasamy, Thamil Selvee; Karunanithi, Puvanan; Naveen, Sangeetha Vasudevaraj; Murali, Malliga Raman; Abbas, Azlina A.; Kamarul, Tunku

    2016-01-01

    Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red

  14. Serum Free Light Chains

    MedlinePlus

    ... and services. Advertising & Sponsorship: Policy | Opportunities Serum Free Light Chains Share this page: Was this page helpful? Also known as: Free Light Chains; SFLC; FLC; Kappa and Lambda Free Light ...

  15. Influences of the recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells in serum-free culture.

    PubMed

    Ishii-Watabe, Akiko; Kanayasu-Toyoda, Toshie; Suzuki, Takuo; Kobayashi, Tetsu; Yamaguchi, Teruhide; Kawanishi, Toru

    2007-10-01

    To improve the safety of cellular therapy products, it is necessary to establish a serum-free cell culture method that can exclude animal-derived materials in order to avoid contamination with transmissible agents. It would be optimal if the proteins necessary to a serum-free culture could be provided as recombinant proteins. In this study, the influences of recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells (HUVECs) in serum-free culture were examined in comparison with the influence of plasma fibronectin (FN). The recombinant proteins used were Pronectin F (PF), Pronectin F PLUS (PFP), Pronectin L (PL), Retronectin (RN), and Attachin (AN). HUVECs adhered more efficiently on PF or PFP than on FN. No cells adhered on PL. Regarding the VEGF or bFGF-induced cell growth, the cells on PF and PFP proliferated at a similar rate to the cells on FN. RN and AN were less effective in supporting cell growth. Since cell adhesion on PF and PFP induced phosphorylation of focal adhesion kinase, they are thought to activate integrin-mediated intracellular signaling. The cells cultured on PF or PFP were able to produce prostaglandin I(2) or tissue-plasminogen activator in response to thrombin. However, thrombin caused detachment of the cells from PF but not from PFP or FN, meaning that the cells were able to adhere more tightly on PFP or FN than on PF. These data indicate that PFP could be applicable as a substitute for plasma FN.

  16. Growth of recombinant Drosophila melanogaster Schneider 2 cells producing rabies virus glycoprotein in bioreactor employing serum-free medium

    PubMed Central

    Galesi, Adriana L. L.; Aguiar, Marcelo A.; Astray, Renato M.; Augusto, Elisabeth F. P.

    2008-01-01

    Drosophila melanogaster Schneider 2 (S2) cells have been increasingly used as a suitable expression system for the production of different recombinant proteins, and the employment of bioreactors for large-scale culture is an important tool for this purpose. In this work, Drosophila S2 cells producing the rabies virus glycoprotein RVGP were cultivated in bioreactor, employing a serum-free medium, aiming an improvement in cell growth and in glycoprotein production. To overcome cell growth limitation commonly observed in stirred flasks, different experiments in bioreactor were performed, in which some system modifications were carried out to attain the desired goal. The study showed that this cell line is considerably sensitive to hydrodynamic forces, and a high cell density (about 16.0 × 106 cells mL−1) was only obtained when Pluronic F68® percentage was increased to 0.6% (w/v). Despite ammonium concentration affected RVGP production, and also cell growth, an elevated amount of the target protein was obtained, attaining 563 ng 10−7 cells. PMID:19003175

  17. Tetramer-organizing polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.

    PubMed

    Schopfer, Lawrence M; Lockridge, Oksana

    2016-06-01

    Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode polyproline-rich peptides. The function of the polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins.

  18. A defined serum-free medium useful for monitoring anti-melanoma responses induced by dendritic cell immunotherapy.

    PubMed

    Bouwer, Anthea L; Netter, Petra; Kemp, Roslyn A; McLellan, Alexander D

    2010-01-31

    Animal sera provide a non-defined source of nutrients and growth factors for mammalian cell culture. Animal serum supplementation may also introduce experimental artefacts, including immune responses against foreign serum proteins. This artefact is particularly apparent in tumour immunotherapy experiments using dendritic cells (DC) and melanoma cells cultured in fetal calf serum (FCS)-replete media. FCS culture of both DC and melanoma cells significantly enhanced anti-tumour responses in mice immunized with DC that had not been pulsed with tumour antigen. Although serum-free media (SFM) may be used for short term culture of cells, most SFM do not support long term culture of tumour cell lines. In addition, in vivo propagation and re-isolation of tumour cells from rodents is expensive, time consuming and only low numbers of viable tumour cells can be recovered from solid tumours. We show that a defined SFM medium is ideal for routine culture of B16 for use in prophylactic DC immunizations, negating the need for in vivo propagation of tumours to avoid FCS effects in tumour implantation experiments.

  19. Stable, position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures.

    PubMed

    Paulsen, D F; Solursh, M; Langille, R M; Pang, L; Chen, W D

    1994-03-01

    Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA's effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21-22 and 23-24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA's effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.

  20. Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening

    PubMed Central

    Hoesli, Corinne A.; Johnson, James D.; Piret, James M.

    2012-01-01

    The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells. PMID:22442738

  1. Serum-free isolation and culture system to enhance the proliferation and bone regeneration of adipose tissue-derived mesenchymal stem cells.

    PubMed

    Sato, Kazutoshi; Itoh, Takehiro; Kato, Toshiki; Kitamura, Yukiko; Kaul, Sunil C; Wadhwa, Renu; Sato, Fujio; Ohneda, Osamu

    2015-05-01

    Cell therapy using human mesenchymal stem cells (MSCs) is an attractive approach for many refractory diseases. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are considered as a favorable tool due to its abundance in the body, easy proliferation, and high cytokine production potency. In order to avoid the risks associated with the use of fetal bovine serum (FBS) in culture that includes batch variations and contamination with pathogens, development of serum-free culture system has been initiated. We have formulated a completely serum-free culture medium (SFM) that could be used not only for the expansion of AT-MSCs but also for initial isolation. We demonstrate that the AT-MSCs isolated and cultured in serum-free medium (AT-MSCs/SFM) possess high proliferation capacity and differentiation potency to osteoblast, adipocyte, and chondrocyte lineages in vitro. In in vivo bone fraction model analysis, AT-MSCs/SFM showed higher bone repair potency and quality of the regenerated bone than the cells cultured in serum-containing medium (AT-MSCs/SCM). This was attributed to the (i) presence of translated cells in the bone, as evidenced by in vivo imaging of the illuminated translated cells and (ii) high level of expression and induction capacity of AT-MSCs/SFM for cytokine BMP2, CCL2, and CCL5. Taken together, we report a new serum-free culture system for AT-MSCs that is suitable for cell therapy.

  2. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers.

  3. Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

    PubMed

    Shaw, J E; Baglia, L A; Leung, K

    1988-09-01

    The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.

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

    PubMed

    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.

  5. Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum-free medium.

    PubMed

    Freerksen, D L; Schroedl, N A; Hartzell, C R

    1984-08-01

    A serum-free, hormone-supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum-supplemented media. To quantitatively compare cell behavior in SFHM with serum-supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+-linked sn-glycerol-3-phosphate dehydrogenase, malate dehydrogenase, NAD+-linked isocitrate dehydrogenase, NADH-cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum-supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: 1) many enzyme activities that may represent dedifferentiation are elevated by serum; 2) NAD-linked glycerol-3-phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; 3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; 4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 micrograms/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.

  6. Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions.

    PubMed

    Jaquet, P; Gunz, G; Grisoli, F

    1985-01-01

    Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH

  7. High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules

    PubMed Central

    Marenah, Lamin; McCahill, Angela; Condie, Alison; Cowan, Scott

    2016-01-01

    This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000–200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%–95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs. Significance This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In

  8. High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules.

    PubMed

    Olivier, Emmanuel N; Marenah, Lamin; McCahill, Angela; Condie, Alison; Cowan, Scott; Mountford, Joanne C

    2016-10-01

    : This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000-200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%-95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs. This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red

  9. A chimeric vitronectin: IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells.

    PubMed

    Manton, Kerry J; Richards, Sean; Van Lonkhuyzen, Derek; Cormack, Luke; Leavesley, David; Upton, Zee

    2010-09-01

    The therapeutic use of human embryonic stem (hES) cells is severely limited by safety concerns regarding their culture in media containing animal-derived or nondefined factors and on animal-derived feeder cells. Thus, there is a pressing need to develop culture techniques that are xeno-free, fully defined, and synthetic. Our laboratory has discovered that insulin-like growth factor (IGF) and vitronectin (VN) bind to each other resulting in synergistic short-term functional effects in several cell types, including keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric VN:IGF-I protein that functionally mimics the effects obtained upon binding of IGF-I to VN. The aim of the current study was to determine whether hES cells can be serially propagated in feeder-cell-free and serum-free conditions using medium containing our novel chimeric VN:IGF-I protein. Here we demonstrate that hES cells can be serially propagated and retain their undifferentiated state in vitro for up to 35 passages in our feeder-cell-free, serum-free, chemically defined media. We have utilized real-time polymerase chain reaction (PCR), immunofluorescence, and fluorescence-activated cell sorter (FACS) analysis to show that the hES cells have maintained an undifferentiated phenotype. In vitro differentiation assays demonstrated that the hES cells retain their pluripotent potential and the karyotype of the hES cells remains unchanged. This study demonstrates that the novel, fully defined, synthetic VN:IGF-I chimera-containing medium described herein is a viable alternative to media containing serum, and that in conjunction with laminin-coated plates facilitates feeder-cell-free and serum-free growth of hES.

  10. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    PubMed

    Hansen, Charlotte Toftmann

    2016-06-01

    Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed.

  11. Serum-Free Medium and Mesenchymal Stromal Cells Enhance Functionality and Stabilize Integrity of Rat Hepatocyte Spheroids

    PubMed Central

    Bao, Ji; Fisher, James E.; Lillegard, Joseph B.; Wang, William; Amiot, Bruce; Yu, Yue; Dietz, Allan B.; Nahmias, Yaakov; Nyberg, Scott L.

    2013-01-01

    Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver. PMID:23006214

  12. Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media.

    PubMed

    Danielpour, D; Riss, T L; Ogasawara, M; Sirbasku, D A

    1988-01-01

    Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4, insulin 10 micrograms/ml, transferrin 10 micrograms/ml, sodium selenite 10 ng/ml, triiodo-L-thyronine 0.3 nM, phosphoethanolamine 5 microM, epidermal growth factor (20 ng/ml), 17 beta-estradiol 2 nM, and bovine serum albumin 20 micrograms/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 microM), glutathione (20 micrograms/ml), and linoleic acid/bovine serum albumin (150 micrograms/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.

  13. High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

    PubMed

    Gaillet, Bruno; Gilbert, Rénald; Amziani, Rachid; Guilbault, Claire; Gadoury, Christine; Caron, Antoine W; Mullick, Alaka; Garnier, Alain; Massie, Bernard

    2007-01-01

    To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium. To increase the transduction level of AdVs, we have also generated a cell line (CHO-cTA-CAR) that expresses additional amounts of the coxackievirus and adenovirus receptor (CAR) on its surface. Recombinant protein production was tested using an AdV carrying the secreted alkaline phosphatase (SEAP) under the control of the CR5 promoter, which is strongly and specifically activated by binding to cTA. The SEAP expression was linked to the expression of the green fluorescent protein (GFP) through an internal ribosome entry site (IRES) to facilitate titration of the AdV. We monitored SEAP expression on a daily basis for 9 days after transduction of CHO-cTA and CHO-cTA-CAR using different quantities of AdVs at 37 and 30 degrees C. Incubation at the latter temperature increased the production of SEAP at least 10-fold, and the presence of CAR increased the transduction level of the AdV. Maximum SEAP production (63 mg/L) was achieved at 6-7 days post-infection at 30 degrees C by transducing CHO-cTA-CAR with 500 infectious particles/cell. Because numerous AdVs can now be generated within a few weeks and large-scale production of AdVs is now a routine procedure, this system could be used to produce rapidly milligram quantities of a battery of recombinant proteins as well as for large-scale protein production.

  14. The effects of hypoxia and serum-free conditions on the stemness properties of human adipose-derived stem cells.

    PubMed

    Wan Safwani, Wan Kamarul Zaman; Wong, Chin Wei; Yong, Kar Wey; Choi, Jane Ru; Mat Adenan, Noor Azmi; Omar, Siti Zawiah; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda

    2016-10-01

    The need to have a better and safer culture condition for expansion of human mesenchymal stem cells (MSCs) is crucial particularly to prevent infection and immune rejection. This is normally associated with the use of animal-based serum in the culture media for cell expansion. The aim of this study is to investigate alternative culture conditions which may provide better and safer environment for cell growth. In the present study, human adipose-derived stem cells (ASCs) at passage 3 were subjected to treatment in 4 conditions: (1) 21 % O2 with fetal bovine serum (FBS), (2) 21 % O2 without FBS, (3) 2 % O2 with FBS and (4) 2 % O2 without FBS followed by subsequent analysis of their phenotype, viability and functionality. We observed that ASCs cultured in all conditions present no significant phenotypic changes. It was found that ASCs cultured in 2 % O2 without serum showed an increase in viability and growth to a certain extent when compared to those cultured in 21 % O2 without serum. However, ASCs cultured in 2 % O2 without serum displayed a relatively low adipogenic and osteogenic potential. On the other hand, interestingly, there was a positive enhancement in chondrogenic differentiation of ASCs cultured in 21 % O2 without serum. Our findings suggest that different culture conditions may be suitable for different indications. In summary, ASCs cultured in serum-free condition can still survive, proliferate and undergo subsequent adipogenic, osteogenic and chondrogenic differentiation. Therefore, FBS is feasible to be excluded for culture of ASCs, which avoids clinical complications.

  15. MAR characteristic motifs mediate episomal vector in CHO cells.

    PubMed

    Lin, Yan; Li, Zhaoxi; Wang, Tianyun; Wang, Xiaoyin; Wang, Li; Dong, Weihua; Jing, Changqin; Yang, Xianjun

    2015-04-01

    An ideal gene therapy vector should enable persistent transgene expression without limitations in safety and reproducibility. Recent researches' insight into the ability of chromosomal matrix attachment regions (MARs) to mediate episomal maintenance of genetic elements allowed the development of a circular episomal vector. Although a MAR-mediated engineered vector has been developed, little is known on which motifs of MAR confer this function during interaction with the host genome. Here, we report an artificially synthesized DNA fragment containing only characteristic motif sequences that served as an alternative to human beta-interferon matrix attachment region sequence. The potential of the vector to mediate gene transfer in CHO cells was investigated. The short synthetic MAR motifs were found to mediate episomal vector at a low copy number for many generations without integration into the host genome. Higher transgene expression was maintained for at least 4 months. In addition, MAR was maintained episomally and conferred sustained EGFP expression even in nonselective CHO cells. All the results demonstrated that MAR characteristic sequence-based vector can function as stable episomes in CHO cells, supporting long-term and effective transgene expression. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method

    PubMed Central

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook

    2017-01-01

    Background Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Methods Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Results Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34+CD43+ hematopoietic progenitor cells (HPCs) and CD34+CD45+ HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro. Conclusion In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs. PMID:28401100

  17. Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity.

    PubMed

    Mandl, Erik W; van der Veen, Simone W; Verhaar, Jan A N; van Osch, Gerjo J V M

    2002-08-01

    For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to find a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and 11-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.

  18. [Metabolic characteristics of GS-nS0 myeloma cells producing anti-CD25 monoclonal antibody in serum-free culture].

    PubMed

    Zhao, Liang; Fan, Li; Zhang, Xu; Tan, Wensong

    2009-07-01

    As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optimized GS-NS0 cell culture and anti-CD25 monoclonal antibody production processes.

  19. Establishment a CHO Cell Line Expressing Human CD52 Molecule

    PubMed Central

    Kadijeh, Tati; Mahsa, Yazdanpanah-Samani; Amin, Ramezani; Elham, Mahmoudi Maymand; Abbas, Ghaderi

    2016-01-01

    Background: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details Methods: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. Results: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. Conclusion: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids PMID:28070536

  20. Genotoxicity of complex mixtures: CHO cell mutagenicity assay

    SciTech Connect

    Frazier, M.E.; Samuel, J.E.

    1985-02-01

    A Chinese hamster ovary (CHO) mammalian cell assay was used to evaluate the genotoxicity of complex mixtures (synthetic fuels). The genotoxicity (mutagenic potency) of the mixtures increased as the temperature of their boiling range increased. Most of the genotoxicity in the 750/sup 0/F+ boiling-range materials was associated with the neutral polycyclic aromatic hydrocarbon (PAH) fractions. Chemical analysis data indicate that the PAH fractions of high-boiling coal liquids contain a number of known chemical carcinogens, including five- and six-ring polyaromatics (e.g., benzo(a)pyrene) as well as four- and five-ring alkyl-substituted PAH (e.g., methylchrysene and dimethylbenzanthracenes); concentrations are a function of boiling point (bp). In vitro genotoxicity was also detected in fractions of nitrogen-containing polyaromatic compounds, as well as in those with aliphatics of hydroxy-containing PAH. Mutagenic activity of some fractions was detectable in the CHO assay in the absence of an exogenous metabolic activation system; in some instances, addition of exogenous enzymes and cofactors inhibited expression of the direct-acting mutagenic potential of the fraction. These data indicate that the organic matrix of the chemical fraction determines whether, and to what degree, various mutagens are expressed in the CHO assay. Therefore, the results of biological assays of these mixtures must be correlated with chemical analyses for proper interpretation of these data. 29 references, 16 figures, 4 tables.

  1. Culture temperature modulates aggregation of recombinant antibody in cho cells.

    PubMed

    Gomez, Natalia; Subramanian, Jayashree; Ouyang, Jun; Nguyen, Mary D H; Hutchinson, Matthew; Sharma, Vikas K; Lin, Andy A; Yuk, Inn H

    2012-01-01

    During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

  2. The effect of growth factor supplementation on corneal stromal cell phenotype in vitro using a serum-free media.

    PubMed

    Lynch, Amy P; O'Sullivan, Finbarr; Ahearne, Mark

    2016-10-01

    In order to expand cells quickly and in high numbers for corneal tissue engineering applications corneal stromal cells, or keratocytes, are often cultured in the presence of serum. However, keratocytes become fibroblastic when exposed to serum leading to a downregulation of corneal stromal specific markers. The purpose of this current study was to determine if corneal stromal cells, made fibroblastic by serum, could display native quiescent keratocyte characteristics when cultured under serum-free conditions supplemented by different growth factors. Markers specific to a native keratocyte phenotype such as keratocan and aldehyde dehydrogenase 3A1 (ALDH3A1) and those specific to a fibrotic phenotype such as α-smooth muscle actin (αSMA) and collagen type III were examined. Cells were cultured in monolayer, self-assembled pellets or collagen hydrogels. Growth factors known to modulate keratocyte phenotype were chosen to supplement the serum free media, specifically insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 and 3 (Tβ1 and Tβ3). The effects of serum-free media, growth factors and culture system on cell proliferation and morphology and extracellular matrix (ECM) synthesis were evaluated. The expression of keratocyte markers was evaluated by real-time PCR, immunofluorescent staining and western blotting. In addition, cell migration was tested using scratch assays. When serum was removed from the cells they displayed a reduction in proliferation and ECM synthesis (not significant), in addition to a significant decrease in migratory capacity (p < 0.05). Serum-free media promoted increased expression of keratocan (130.68 ± 47.44-fold increase; p < 0.05) and collagen type I (15.58 ± 9.49-fold increase; p < 0.05). However, there was no significant change in ALDH3A1 and αSMA expression, while collagen type III expression was significantly increased (44.66 ± 25.61-fold increase; p < 0.05). In addition, cells retained an

  3. Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

    PubMed

    Klein, Tino; Heremans, Yves; Heimberg, Harry; Pipeleers, Daniel; Madsen, Ole D; Serup, Palle; Heller, R Scott

    2009-01-01

    We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.

  4. One-step induction of neurons from mouse embryonic stem cells in serum-free media containing vitamin B12 and heparin.

    PubMed

    Yamazoe, Hironori; Kobori, Masato; Murakami, Yoshinobu; Yano, Keiichi; Satoh, Mitsuo; Mizuseki, Kenji; Sasai, Yoshiki; Iwata, Hiroo

    2006-01-01

    We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, beta-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5-fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

  5. Rapid cryopreservation of five mammalian and one mosquito cell line at -80 degrees C while attached to flasks in a serum free cryopreservative.

    PubMed

    Corsini, Joe; Mann, Ethan

    2005-01-01

    Cell culturing, and the requisite storage of cell lines at ultra-low temperatures, is used in most laboratories studying or using eukaryotic proteomics, genomics, microarray, and RNA technologies. In this study we have observed that A72(dog), CRFK(cat), NB324K(human), MCF7(human), WI38(human), and C636(mosquito) cells were effectively cryopreserved at -80 degrees C while attached to the substratum of 25 cm2 tissue culture flasks. This was accomplished using a serum free crypreservative recently developed by Corsini and co-workers. The technique allows for significant savings of time and money in laboratories that rapidly process numerous cell lines.

  6. Characterization of Neurogenic Potential of Dental Pulp Stem Cells Cultured in Xeno/Serum-Free Condition: In Vitro and In Vivo Assessment

    PubMed Central

    Jung, Jieun; Kim, Jong-Wan; Moon, Ho-Jin; Hong, Jin Young

    2016-01-01

    Neural stem cells (NSCs) have a high potency for differentiation to neurons and glial cells for replacement of damaged cells and paracrine effects for the regeneration and remyelination of host axons. Dental pulp is known to have a potential to differentiate into neural-like cells; therefore, dental pulp may be used as an autologous cell source for neural repair. In this study, we selectively expanded stem cells from human dental pulp in an initial culture using NSC media under xeno- and serum-free conditions. At the initial step of primary culture, human dental pulp was divided into two groups according to the culture media: 10% fetal bovine serum medium group (FBS group) and NSC culture medium group (NSC group). In the NSC group relative to the FBS group, the expression of NSC markers and the concentrations of leukemia inhibitory factor, nerve growth factor, and stem cell factor were higher, although their expression levels were lower than those of human fetal NSCs. The transplanted cells of the NSC group survived well within the normal brain and injured spinal cord of rats and expressed nestin and Sox2. Under the xeno- and serum-free conditions, autologous human dental pulp-derived stem cells might prove useful for clinical cell-based therapies to repair damaged neural tissues. PMID:27688776

  7. Serum-free culture of an embryonic cell line from Bombyx mori and reinforcement of susceptibility of a recombinant BmNPV by cooling.

    PubMed

    Imanishi, Shigeo; Kobayashi, Jun; Sekine, Toshiaki

    2012-03-01

    We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67±5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.

  8. Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

    PubMed

    Zhao, Peng; Fu, Juanling; Yao, Biyun; Jia, Yongrui; Zhang, Hongtao; Li, Xuehui; Dong, Lisha; Gao, Ya; Liu, Wenli; Chen, Wen; Zhou, Zongcan

    2016-02-05

    To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis.

  9. What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

    PubMed

    Nakashima, Yoshiki; Omasa, Takeshi

    2016-01-01

    Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell-cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new scaffold for hi

  10. Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2

    PubMed Central

    TSUGENO, YUTA; SATO, FUYUKI; MURAGAKI, YASUTERU; KATO, YUKIO

    2014-01-01

    The majority of cells are cultured with Dulbecco’s modified Eagle’s medium (DMEM) or RPMI supplemented with fetal bovine serum (FBS), which contains numerous factors, including cytokines, nutrients and unknown growth factors. These factors may affect cell growth, apoptosis and differentiation. The serum-free medium, STK2, has been previously reported as suitable for the cell culture of human mesenchymal stem cells. However, how STK1 or STK2 affect the cell proliferation of normal and cancer cells remains unknown. The present study examined the growth of the human gingival fibroblast (HGF-1) cell-line and the HSC-3, CA9-22 and MSTO cancer cell-lines, cultured with STK1 and STK2. STK1 increased the cell proliferation of HGF-1 compared to DMEM by assessment with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay, whereas STK1 and STK2 markedly inhibited the cell proliferation of HSC-3 and MSTO. The cell proliferation rate of CA9-22 cultured with STK1 or STK2 for 96 h was ~2-fold higher than the rate for 24 h culture. The shape of the HSC-3 cells was also found to have changed to round when cultured with STK2. These results indicate that STK1 increased the cell proliferation of HGF-1 compared to DMEM, whereas the proliferation of HSC-3 and MSTO was inhibited by STK1 and STK2. Thus, STK1 and STK2 had different affects on the cell growth of HGF-1, CA9-22, HSC-3 and MSTO. PMID:25054004

  11. Cell culture of human gingival fibroblasts, oral cancer cells and mesothelioma cells with serum-free media, STK1 and STK2.

    PubMed

    Tsugeno, Yuta; Sato, Fuyuki; Muragaki, Yasuteru; Kato, Yukio

    2014-09-01

    The majority of cells are cultured with Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with fetal bovine serum (FBS), which contains numerous factors, including cytokines, nutrients and unknown growth factors. These factors may affect cell growth, apoptosis and differentiation. The serum-free medium, STK2, has been previously reported as suitable for the cell culture of human mesenchymal stem cells. However, how STK1 or STK2 affect the cell proliferation of normal and cancer cells remains unknown. The present study examined the growth of the human gingival fibroblast (HGF-1) cell-line and the HSC-3, CA9-22 and MSTO cancer cell-lines, cultured with STK1 and STK2. STK1 increased the cell proliferation of HGF-1 compared to DMEM by assessment with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay, whereas STK1 and STK2 markedly inhibited the cell proliferation of HSC-3 and MSTO. The cell proliferation rate of CA9-22 cultured with STK1 or STK2 for 96 h was ~2-fold higher than the rate for 24 h culture. The shape of the HSC-3 cells was also found to have changed to round when cultured with STK2. These results indicate that STK1 increased the cell proliferation of HGF-1 compared to DMEM, whereas the proliferation of HSC-3 and MSTO was inhibited by STK1 and STK2. Thus, STK1 and STK2 had different affects on the cell growth of HGF-1, CA9-22, HSC-3 and MSTO.

  12. Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium.

    PubMed Central

    Tres, L L; Kierszenbaum, A L

    1983-01-01

    Spermatogenic cells from 20- to 35-day-old rats were grown in vitro in the presence of Sertoli cells maintained in serum-free hormone/growth factor-supplemented medium and alternating high/low concentrations of follicle-stimulating hormone in the medium. In cell reaggregation experiments, spermatogenic cells reassociate with Sertoli cells but not with peritubular cells or cell-free substrate. Autoradiographic experiments using [3H]thymidine as a labeled precursor for DNA synthesis show that spermatogonia and preleptotene spermatocytes, connected by cytoplasmic bridges, have a synchronous S phase. [3H]Thymidine-labeled preleptotene spermatocytes progress until later stages of meiotic prophase. Time-lapse cinematographic studies of Sertoli/spermatogenic cell cocultures show three major movement patterns. While Sertoli cell cytoplasmic processes between adjacent cells display tensional forces, spermatogonia are engaged in oscillatory cell movements different from the nuclear rotation observed in meiotic prophase spermatocytes. Results of this study show that the proliferation of premeiotic cells and the differentiation of meiotic prophase cells do occur in vitro in association with Sertoli cells maintained in a medium that allows differentiated cell functions. Images PMID:6407012

  13. Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

    PubMed

    Romand, Sandrine; Jostock, Thomas; Fornaro, Mara; Schmidt, Joerg; Ritter, Anett; Wilms, Burkhard; Laux, Holger

    2016-05-01

    Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells.

  14. Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media.

    PubMed

    Alfred, Roz; Taiani, Jaymi T; Krawetz, Roman J; Yamashita, Akihiro; Rancourt, Derrick E; Kallos, Michael S

    2011-09-01

    The generation of tissue-engineered constructs from stem cells for the treatment of musculoskeletal diseases may have immense impact in regenerative medicine, but there are difficulties associated with stem cell culture and differentiation, including the use of serum. Here we present serum-free protocols for the successful production of murine embryonic stem cell (mESC) derived osteoblasts and chondrocytes on CultiSpher S macroporous microcarriers in stirred suspension bioreactors. Various inoculum forms and agitation rates were investigated. Produced osteogenic cells were implanted ectopically into SCID mice and orthotopically into a murine burr-hole fracture model. Osterix, osteocalcin and collagen type I were upregulated in osteogenic cultures, while aggrecan and collagen type II were upregulated in chondrogenic cultures. Histological analysis using alizarin red S, von Kossa and alcian blue staining confirmed the presence of osteoblasts and chondrocytes, respectively in cultured microcarriers and excised tissue. Finally, implantation of derived cells into a mouse fracture model revealed cellular integration without any tumor formation. Overall, microcarriers may provide a supportive scaffold for ESC expansion and differentiation in a serum-free bioprocess for in vivo implantation. These findings lay the groundwork for the development of clinical therapies for musculoskeletal injuries and diseases using hESCs and iPS cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.

    PubMed

    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.

  16. Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

    SciTech Connect

    Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S. ); Li, S.A.; Li, J.J. )

    1989-03-01

    It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17{beta}-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17{beta}-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17{beta}-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17{beta}-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17{beta}-estradiol, ({sup 3}H)thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney.

  17. The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2015-04-10

    Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

  18. Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

    PubMed Central

    Gervais-St-Amour, Catherine

    2016-01-01

    The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC) from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50%) when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium. PMID:27872867

  19. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    PubMed Central

    Pilbrough, Warren; Munro, Trent P.; Gray, Peter

    2009-01-01

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  20. Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

    PubMed

    Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

    2013-01-01

    The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

  1. Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

    PubMed

    Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

    2017-03-15

    Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.

  2. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  3. Efficient neural differentiation of mouse pluripotent stem cells in a serum-free medium and development of a novel strategy for enrichment of neural cells.

    PubMed

    Verma, Isha; Rashid, Zubin; Sikdar, Sujit K; Seshagiri, Polani B

    2017-10-01

    Pluripotent stem cells (PSCs) offer an excellent model to study neural development and function. Although various protocols have been developed to direct the differentiation of PSCs into desired neural cell types, many of them suffer from limitations including low efficiency, long duration of culture, and the use of expensive, labile, and undefined growth supplements. In this study, we achieved efficient differentiation of mouse PSCs to neural lineage, in the absence of exogenous molecules, by employing a serum-free culture medium containing knockout serum replacement (KSR). Embryoid bodies (EBs) cultured in this medium predominantly produced neural cells which included neural progenitors (15-18%), immature neurons (8-24%), mature neurons (10-26%), astrocytes (27-61%), and oligodendrocytes (∼1%). Different neuronal subtypes including glutamatergic, GABAergic, cholinergic, serotonergic, and dopaminergic neurons were generated. Importantly, neurons generated in the KSR medium were electrically active. Further, the EB scooping strategy, involving the removal of the EB core region from the peripheral EB outgrowth, resulted in the enrichment of PSC-derived neural cells. Taken together, this study provides the evidence that the KSR medium is ideal for the rapid and efficient generation of neural cells, including functional neurons, from PSCs without the requirement of any other additional molecule. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

  4. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells

    PubMed Central

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  5. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells.

    PubMed

    Goh, John S Y; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei

    2014-01-01

    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.

  6. A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces.

    PubMed

    Tátrai, Péter; Sági, Bernadett; Szigeti, Anna; Szepesi, Aron; Szabó, Ildikó; Bősze, Szilvia; Kristóf, Zoltán; Markó, Károly; Szakács, Gergely; Urbán, István; Mező, Gábor; Uher, Ferenc; Német, Katalin

    2013-02-01

    Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Ser(i)-DL-Ala(m))] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss(®) bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.

  7. [COMPARATIVE CYTOGENETIC ANALYSIS OF MONOLAYER AND SUSPENSION CHINESE HAMSTER OVARY CELL LINES CHO(dhfr-)].

    PubMed

    Stefanova, V N; Yartseva, N M; Petrov, A V

    2015-01-01

    The karyotypes of CHO(dhfr-) and CHO(dhfr-)/susp Chinese hamster ovary cell lines were investigated with the use of GTG-staining. Modal chromosome set consists of 20 and 18 chromosomes respectively. The karyotypes of both cell lines were stable with constant ratio of normal chromosomes and chromosomes with structural rearrangements. Monosomy for chromosomes 1, 2, 4, 5, 8 was observed in both cell lines and for chromosome 9 in CHO(dhfr-)/susp cell line. The differences between CHO(dhfr-) cell lines studied by us consists of inclusion of part of chromosome 7 in der(6)t(1;6), rearrangement of del(5) and monosomy of chromosome 9. It was shown that in karyotypes of all CHO cell lines studied up today there are 5 common structurally chromosome rearrangements: del(2), inv(3), add(6), del(9) and mar1. In both CHO(dhfr-) cell lines investigated by us three unique chromosome rearrangements: del(1), der(6)t(1,6) and mar3 were revealed. Necessity of simultaneous GTG and FISH analysis of chromosomes rearrangements in the CHO cell lines under study is discussed.

  8. Fetal rat lung type II cell differentiation in serum-free isolated cell culture: modulation and inhibition.

    PubMed

    Fraslon, C; Lacaze-Masmonteil, T; Zupan, V; Chailley-Heu, B; Bourbon, J R

    1993-05-01

    Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

  9. Human mesenchymal stem cells possess different biological characteristics but do not change their therapeutic potential when cultured in serum free medium.

    PubMed

    Wang, Youwei; Wu, Hehe; Yang, Zhouxin; Chi, Ying; Meng, Lei; Mao, Aibin; Yan, Shulin; Hu, Shanshan; Zhang, Jianzhong; Zhang, Yun; Yu, Wenbo; Ma, Yue; Li, Tao; Cheng, Yan; Wang, Yongjuan; Wang, Shanshan; Liu, Jing; Han, Jingwen; Li, Caiyun; Liu, Li; Xu, Jian; Han, Zhi Bo; Han, Zhong Chao

    2014-12-04

    Mesenchymal stem cells (MSCs) are widely investigated in clinical researches to treat various diseases. Classic culture medium for MSCs, even for clinical use, contains fetal bovine serum. The serum-containing medium (SCM) seems a major obstacle for MSCs-related therapies due to the risk of contamination of infectious pathogens. Some studies showed that MSCs could be expanded in serum free medium (SFM); however, whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in a chemical defined serum free medium. Growth, multipotency, surface antigen expression, telomerase, immunosuppressive ability, gene expression profile and genomic stability of hUC-MSCs cultured in SFM and SCM were analyzed and compared side by side. hUC-MSCs propagated more slowly and senesce ultimately in SFM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in growth rate, telomerase, gene expression profile. However, SFM-expanded hUC-MSCs maintained multipotency and the profile of surface antigen which were used to define human MSCs. Both SFM- and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture. hUC-MCSs could be expanded in SFM safely to obtain enough cells for clinical application, meeting the basic criteria for human mesenchymal stem cells. hUC-MSCs cultured in SFM were distinct from hUC-MSCs cultured in SCM, yet they remained therapeutic potentials for future regenerative medicine.

  10. A new clinical-scale serum-free xeno-free medium efficient in ex vivo amplification of mesenchymal stromal cells does not support mesenchymal stem cells.

    PubMed

    Gerby, Sandie; Attebi, Esther; Vlaski, Marija; Ivanovic, Zoran

    2017-02-01

    We evaluated a new serum-free, xeno-free medium (Xuri, GE HealthCare) in ex vivo cultures for amplification of mesenchymal stromal cells (MStroC) in comparison with classical culture supplemented with fetal calf serum and basic fibroblast growth factor. MStroC and mesenchymal stem cell (MSC) proliferative capacities were studied in bulk cultures and single-cell cultures with assay of secondary replating capacity of individual clones. Flow-cytometric phenotype analysis and proliferative history analysis were also performed. In cultures initiated with previously amplified and cryopreserved MStroC from human marrow, Xuri medium enabled a total cell expansion fold comparable to one obtained in control fetal calf serum (FCS)-supplemented culture. However, both the number and the proliferative capacity of colony-forming unit-fibroblast were greatly reduced in Xuri medium cultures. This is even more evident in single-cell cultures, where, in rare positive wells, only several cells were found in Xuri cultures, compared to abundant cell content in FCS and α-minimal essential medium cultures. Replating these single-cell clones in secondary cultures (FCS in both cases) revealed a total exhaustion of MSC proliferative capacity after Xuri primary culture. Since in both conditions after a 7-day bulk culture, similar immunophenotype and proliferative history were found when the standard MSC immunophenotype panel was employed, the loss of proliferative capacity in Xuri medium shows that it cannot maintain functional MSC population. This is a drastic example showing that the real MSC activity can be completely unrelated to the immunophenotype considered as MSC phenotype. © 2016 AABB.

  11. Corneal keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with FGF-2, TGF-ß3 and retinoic acid.

    PubMed

    Sidney, Laura E; Hopkinson, Andrew

    2016-09-29

    Keratocytes of the corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a corneal MSC (cMSC) phenotype is achieved, regaining the keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of keratocytes to cMSC and compared different supplements in their ability to return cMSC to a keratocyte phenotype. Immunofluorescence and RT-qPCR demonstrated in vivo keratocyte expression of ALDH3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not keratocyte markers. Differentiating the cMSC back to a keratocyte phenotype using non-supplemented, serum-free medium restored keratocyte markers but did not maintain cell viability or support corneal extracellular matrix (ECM) production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and retinoic acid (RA) maintained viability, restored expression of CD34, ALDH3A1 and keratocan, and facilitated production of abundant ECM as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with RA, FGF-2 and TGF-β3 can enhance this effect. This is relevant for development of corneal regenerative strategies that require the production of a keratocyte phenotype.

  12. Alteration of histone acetylation pattern during long-term serum-free culture conditions of human fetal placental mesenchymal stem cells.

    PubMed

    Zhu, Yongzhao; Song, Xumei; Han, Fei; Li, Yukui; Wei, Jun; Liu, Xiaoming

    2015-01-01

    Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.

  13. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  14. CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives.

    PubMed

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E; Faustrup Kildegaard, Helene

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome-wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9-mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Controllability Analysis of Protein Glycosylation in Cho Cells

    PubMed Central

    St. Amand, Melissa M.; Tran, Kevin; Radhakrishnan, Devesh; Robinson, Anne S.; Ogunnaike, Babatunde A.

    2014-01-01

    To function as intended in vivo, a majority of biopharmaceuticals require specific glycan distributions. However, achieving a precise glycan distribution during manufacturing can be challenging because glycosylation is a non-template driven cellular process, with the potential for significant uncontrolled variability in glycan distributions. As important as the glycan distribution is to the end-use performance of biopharmaceuticals, to date, no strategy exists for controlling glycosylation on-line. However, before expending the significant amount of effort and expense required to develop and implement on-line control strategies to address the problem of glycosylation heterogeneity, it is imperative to assess first the extent to which the very complex process of glycosylation is controllable, thereby establishing what is theoretically achievable prior to any experimental attempts. In this work, we present a novel methodology for assessing the output controllability of glycosylation, a prototypical example of an extremely high-dimensional and very non-linear system. We first discuss a method for obtaining the process gain matrix for glycosylation that involves performing model simulations and data analysis systematically and judiciously according to a statistical design of experiments (DOE) scheme and then employing Analysis of Variance (ANOVA) to determine the elements of process gain matrix from the resulting simulation data. We then discuss how to use the resulting high-dimensional gain matrix to assess controllability. The utility of this method is demonstrated with a practical example where we assess the controllability of various classes of glycans and of specific glycoforms that are typically found in recombinant biologics produced with Chinese Hamster Ovary (CHO) cells. In addition to providing useful insight into the extent to which on-line glycosylation control is achievable in actual manufacturing processes, the results also have important implications for

  16. The hollow fiber bioreactor as a stroma-supported, serum-free ex vivo expansion platform for human umbilical cord blood cells.

    PubMed

    Xue, Cao; Kwek, Kenneth Y C; Chan, Jerry K Y; Chen, Qingfeng; Lim, Mayasari

    2014-07-01

    The bone marrow microenvironment plays an integral role in the regulation of hematopoiesis. Residing stromal cells and the extracellular matrix in the bone marrow microenvironment provide biological signals that control hematopoietic stem cell (HSC) function. In this study, we developed a bio-mimetic co-culture platform using the hollow fiber bioreactor (HFBR) for ex vivo expansion of HSCs. We evaluated the efficacy of such a platform in comparison to standard cultures performed on tissue culture polystyrene (TCP), using a human stromal cell line (HS-5) as stromal support, co-cultured with lineage-depleted human cord blood cells in serum-free medium supplemented with a cytokine cocktail. Our results showed that the performance of the HFBR in supporting total cell and CD34(+) progenitor cell expansion was comparable to that of cultures on TCP. Cells harvested from the HFBR had a higher clonogenic ability. The performance of ex vivo-expanded cells from the HFBR in hematopoietic reconstitution in humanized mice was comparable to that of the TCP control. Scanning electron microscopy revealed that stroma cell growth inside the HFBR created a three-dimensional cell matrix architecture. These findings demonstrate the feasibility of utilizing the HFBR for creating a complex cell matrix architecture, which may provide good in vitro mimicry of the bone marrow, supporting large-scale expansion of HSCs.

  17. Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

    SciTech Connect

    Jozan, S.; Faye, J.C.; Tournier, J.F.; Tauber, J.P.; David, J.F.; Bayard, F.

    1985-11-27

    The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures (''confluent-log'' cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that ''confluent-log'' cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.

  18. Sub-micron and nanoscale feature depth modulates alignment of stromal fibroblasts and corneal epithelial cells in serum-rich and serum-free media.

    PubMed

    Fraser, Sarah A; Ting, Yuk-Hong; Mallon, Kelly S; Wendt, Amy E; Murphy, Christopher J; Nealey, Paul F

    2008-09-01

    Topographic features are generally accepted as being capable of modulating cell alignment. Of particular interest is the potential that topographic feature geometry induces cell alignment indirectly through impacting adsorbed proteins from the cell culture medium on the surface of the substrate. However, it has also been reported that micron-scale feature depth significantly impacts the level of alignment of cellular populations on topography, despite being orders of magnitude larger than the average adsorbed protein layer (nm). In order to better determine the impact of biomimetic length scale topography and adsorbed protein interaction on cellular morphology we have systematically investigated the effect of combinations of sub-micron to nanoscale feature depth and lateral pitch on corneal epithelial cell alignment. In addition we have used the unique properties of a serum-free media alternative in direct comparison to serum-rich medium to investigate the role of culture medium protein composition on cellular alignment to topographically patterned surfaces. Our observation that increasing groove depth elicited larger populations of corneal epithelial cells to align regardless of culture medium composition and of cell orientation with respect to the topography, suggests that these cells can sense changes in topographic feature depths independent of adsorbed proteins localized along ridge edges and tops. However, our data also suggests a strong combinatory effect of topography with culture medium composition, and also a cell type dependency in determining the level of cell elongation and alignment to nanoscale topographic features.

  19. Sub-micron and nanoscale feature depth modulates alignment of stromal fibroblasts and corneal epithelial cells in serum-rich and serum-free media

    PubMed Central

    Fraser, Sarah A.; Ting, Yuk-Hong; Mallon, Kelly S.; Wendt, Amy E.; Murphy, Christopher J.; Nealey, Paul F.

    2011-01-01

    Topographic features are generally accepted as being capable of modulating cell alignment. Of particular interest is the potential that topographic feature geometry induces cell alignment indirectly through impacting adsorbed proteins from the cell culture medium on the surface of the substrate. However, it has also been reported that micron-scale feature depth significantly impacts the level of alignment of cellular populations on topography, despite being orders of magnitude larger than the average adsorbed protein layer (nm). In order to better determine the impact of biomimetic length scale topography and adsorbed protein interaction on cellular morphology we have systematically investigated the effect of combinations of sub-micron to nanoscale feature depth and lateral pitch on corneal epithelial cell alignment. In addition we have used the unique properties of a serum-free media alternative in direct comparison to serum-rich medium to investigate the role of culture medium protein composition on cellular alignment to topographically patterned surfaces. Our observation that increasing groove depth elicited larger populations of corneal epithelial cells to align regardless of culture medium composition and of cell orientation with respect to the topography, suggests that these cells can sense changes in topographic feature depths independent of adsorbed proteins localized along ridge edges and tops. However, our data also suggests a strong combinatory effect of topography with culture medium composition, and also a cell type dependency in determining the level of cell elongation and alignment to nanoscale topographic features. PMID:18041718

  20. A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.

    PubMed

    Miyagiwa, M; Ichida, T; Tokiwa, T; Sato, J; Sasaki, H

    1989-06-01

    A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in RPMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.

  1. Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

    PubMed

    Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

    2010-12-01

    The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

  2. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells.

    PubMed

    da Silva, Cláudia Lobato; Gonçalves, Raquel; Crapnell, Kirsten B; Cabral, Joaquim M S; Zanjani, Esmail D; Almeida-Porada, Graça

    2005-07-01

    We investigated the role of human stromal layers (hu-ST) on the ex vivo expansion/maintenance of human hematopoietic stem/progenitor cells (HSC) from adult bone marrow (BM) and umbilical cord blood (CB). BM and CB CD34(+)-enriched cells were cultured in serum-free medium supplemented with SCF, bFGF, LIF, and Flt-3, in the presence or absence of stroma, and analyzed for proliferation, phenotype, and clonogenic potential. Significant expansion of BM and CB CD34(+) and CD34(+)CD38(-) cells were achieved in the presence of hu-ST. The differentiative potential of both BM and CB CD34(+)-enriched cells cocultured with hu-ST was primarily shifted toward the myeloid lineage, while maintaining/expanding a CD7(+) population. Clonogenic analysis of the expanded cells showed increases in progenitors of the myeloid lineage, including colony-forming unit-granulocyte, macrophage (CFU-GM) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-Mix) for both BM (stroma and stroma-free conditions) and CB cells in the presence of stroma. These results indicate that adult hu-ST in the presence of appropriate cytokines can be used to efficiently expand/maintain myeloid and lymphoid cell populations from human BM and CB HSC.

  3. Cultivation of rat granulosa cells in a serum-free chemically defined medium--a useful model to study lipoprotein metabolism.

    PubMed

    Azhar, S; Tsai, L; Maffe, W; Reaven, E

    1988-11-25

    We have developed a chemically defined, serum-free medium for the culture of rat granulosa cells. This medium contains Dulbecco's modified Eagle's medium/Ham's nutrient F12 (DME:F12) (1:1) plus insulin (2 micrograms/ml), hydrocortisone (100 ng/ml), transferrin (5 micrograms/ml) and fibronectin (2 micrograms/cm2). Granulosa cells grown in this medium have an absolute requirement for added cholesterol-rich lipoproteins for steroidogenesis. When cells are cultured in basal medium, progestin production is low; when cells are cultured in the presence of follicle-stimulating hormone (FSH) or dibutyryl cAMP [Bu)2 cAMP), progestin secretion is increased 10-100-fold. Both heterologous and homologous lipoproteins synergistically increased the effects of (Bu)2 cAMP or FSH: e.g., addition to the medium of human (h)-HDL3 produced a significant increase in both basal (approx. 15-fold) and (Bu)2 cAMP-stimulated (approx. 1000-2000-fold) progestin production. LDL were less effective than HDL at equivalent concentrations of lipoprotein cholesterol. FSH invoked changes similar to that of (Bu)2 cAMP, although the magnitude of the FSH-induced change was less dramatic than that seen with (Bu)2 cAMP. The effect of h-HDL3 and h-LDL on both basal and hormone-stimulated progestin production was concentration- and time-dependent. The maximum effect of h-HDL3 was achieved at a protein concentration of 500 micrograms/ml, with an ED50 of approx. 90 micrograms/ml. In contrast, h-LDL was most effective at a concentration of 30-40 micrograms protein/ml. Likewise, rat (r-)HDL and r-LDL supported steroidogenesis in a concentration-dependent manner. Maximal responses to all additions were observed after 72 h of treatment. Granulosa cells secreted 20 alpha-hydroxypregn-4-ene-3-one as the predominant steroid in response to (Bu)2 cAMP. However, with the addition of h-HDL3, the major secreted product was progesterone. In conclusion, rat granulosa cells maintained in the described serum-free medium are

  4. [Differentiation of C17.2 neural stem cells into neural cells induced by serum-free conditioned medium of olfactory ensheathing cells and cell viability detection of differentiated cells].

    PubMed

    Wang, Lei; Duan, Da; Zhao, Zhenyu; Teng, Xiaohua; Liu, Bo; Ge, Lite; Lu, Ming

    2014-05-01

    To study the possibility of the C17.2 neural stem cells (NSCs) differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells (OECs) and to detect the cell viability of the differentiated cells. OECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining [microtubule-associated protein 2 (MAP-2) and beta-tubulin-III] and Western blot (Nestin, beta-tubulin-III, and MAP-2) were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase (LDH) release in the medium. In the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that beta3-tubulin-III and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of beta-tubulin-III and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group (P < 0.05). The LDH release and the cell viability were 130.60% +/- 6.86% and 62.20% +/- 3.82% in the experimental group, and

  5. Extracellular matrix (ECM) modulates the EGF-induced migration of liver epithelial cells in serum-free, hormone-supplemented medium.

    PubMed

    Bade, E G; Nitzgen, B

    1985-04-01

    The influence of the extracellular matrix (ECM) glycoproteins collagen IV, laminin (LN), and fibronectin (FN) on the in vitro migration of epithelial cells was studied using the ECM migration track method (4) with preparations immunostained for LN and FN. The locomotion of rat liver epithelial cells stimulated to migrate in serum-free medium by epidermal growth factor (EGF) in the presence of insulin is inhibited by substratum-bound FN. The inhibition is concentration-dependent up to 0.7 microgram of the protein per cm2. Neither LN nor collagen IV decreased the number of migrating cells, indicating that the inhibition is a specific effect of fibronectin. The data also indicate that the FN-mediated inhibition of migration is an additional and not alternative mechanism to the well-established contact inhibition of locomotion (1) which also occurs in liver epithelial cell cultures. The system is being used for a further analysis of the factors that influence migration of normal and neoplastic epithelial cells and the biochemical mechanisms underlying the migration reaction.

  6. Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

    PubMed

    Mattos, Diogo A; Silva, Marlon V; Gaspar, Luciane P; Castilho, Leda R

    2015-08-20

    In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.

  7. The GalNAc-type O-Glycoproteome of CHO cells characterized by the SimpleCell strategy.

    PubMed

    Yang, Zhang; Halim, Adnan; Narimatsu, Yoshiki; Jitendra Joshi, Hiren; Steentoft, Catharina; Schjoldager, Katrine Ter-Borch Gram; Alder Schulz, Morten; Sealover, Natalie R; Kayser, Kevin J; Paul Bennett, Eric; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2014-12-01

    The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).

  8. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    PubMed

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  9. Cancer stem cells from human glioblastoma resemble but do not mimic original tumors after in vitro passaging in serum-free media

    PubMed Central

    Esteban-Rubio, Susana; Rackov, Gorjana; Rodríguez-Fanjul, Vanessa; Cruz, Jorge Oliver-De La; Prat-Acín, Ricardo; Peris-Celda, María; Blesa, David; Ramírez-Jiménez, Laura; Sánchez-Gómez, Pilar; Perona, Rosario; Escobedo-Lucea, Carmen; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2016-01-01

    Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas. PMID:27589567

  10. Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

    PubMed Central

    Duinhouwer, Lucia E.; Tüysüz, Nesrin; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J.; ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC. PMID:25807521

  11. Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures.

    PubMed

    Duinhouwer, Lucia E; Tüysüz, Nesrin; Rombouts, Elwin W J C; Ter Borg, Mariette N D; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J; Ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.

  12. Cancer stem cells from human glioblastoma resemble but do not mimic original tumors after in vitro passaging in serum-free media.

    PubMed

    García-Romero, Noemí; González-Tejedo, Carmen; Carrión-Navarro, Josefa; Esteban-Rubio, Susana; Rackov, Gorjana; Rodríguez-Fanjul, Vanessa; Oliver-De La Cruz, Jorge; Prat-Acín, Ricardo; Peris-Celda, María; Blesa, David; Ramírez-Jiménez, Laura; Sánchez-Gómez, Pilar; Perona, Rosario; Escobedo-Lucea, Carmen; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2016-10-04

    Human gliomas harbour cancer stem cells (CSCs) that evolve along the course of the disease, forming highly heterogeneous subpopulations within the tumour mass. These cells possess self-renewal properties and appear to contribute to tumour initiation, metastasis and resistance to therapy. CSC cultures isolated from surgical samples are considered the best preclinical in vitro model for primary human gliomas. However, it is not yet well characterized to which extent their biological and functional properties change during in vitro passaging in the serum-free culture conditions. Here, we demonstrate that our CSC-enriched cultures harboured from one to several CSC clones from the human glioma sample. When xenotransplanted into mouse brain, these cells generated tumours that reproduced at least three different dissemination patterns found in original tumours. Along the passages in culture, CSCs displayed increased expression of stem cell markers, different ratios of chromosomal instability events, and a varied response to drug treatment. Our findings highlight the need for better characterization of CSC-enriched cultures in the context of their evolution in vitro, in order to uncover their full potential as preclinical models in the studies aimed at identifying molecular biomarkers and developing new therapeutic approaches of human gliomas.

  13. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

    PubMed Central

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    Background: The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. Methods: In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. Results: In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. Conclusion: The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells. PMID:26232332

  14. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles.

    PubMed

    Davami, Fatemeh; Eghbalpour, Farnaz; Nematollahi, Leila; Barkhordari, Farzaneh; Mahboudi, Fereidoun

    2015-01-01

    The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

  15. The art of CHO cell engineering: A comprehensive retrospect and future perspectives.

    PubMed

    Fischer, Simon; Handrick, René; Otte, Kerstin

    2015-12-01

    Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future.

  16. TGF β-1 administration during ex vivo expansion of human articular chondrocytes in a serum-free medium redirects the cell phenotype toward hypertrophy.

    PubMed

    Narcisi, R; Quarto, R; Ulivi, V; Muraglia, A; Molfetta, L; Giannoni, P

    2012-09-01

    Cell-based cartilage resurfacing requires ex vivo expansion of autologous articular chondrocytes. Defined culture conditions minimize expansion-dependent phenotypic alterations but maintenance of the cells' differentiation potential must be carefully assessed. Transforming growth factor β-1 (TGF β-1) positively regulates the expression of several cartilage proteins, but its therapeutic application in damaged cartilage is controversial. Thus we evaluated the phenotypic outcomes of cultured human articular chondrocytes exposed to TGF β-1 during monolayer expansion in a serum-free medium. After five doublings cells were transferred to micromass cultures to assess their chondrogenic differentiation, or replated in osteogenic medium. Immunocytostainings of micromasses of TGF-expanded cells showed loss of aggrecan and type II collagen. Positivity was evidenced for RAGE, IHH, type X collagen and for apoptotic cells, paralleling a reduction of BCL-2 levels, suggesting hypertrophic differentiation. TGF β-1-exposed cells also evidenced increased mRNA levels for bone sialoprotein, osteopontin, matrix metalloproteinase-13, TIMP-3, VEGF and SMAD7, enhanced alkaline phosphatase activity and pyrophosphate availability. Conversely, SMAD3 mRNA and protein contents were reduced. After osteogenic induction, only TGF-expanded cells strongly mineralized and impaired p38 kinase activity, a contributor of chondrocytes' differentiation. To evaluate possible endochondral ossification progression, we seeded the chondrocytes on hydroxyapatite scaffolds, subsequently implanted in an in vivo ectopic setting, but cells failed to reach overt ossification; nonetheless, constructs seeded with TGF-exposed cells displayed blood vessels of the host vascular supply with enlarged diameters, suggestive of vascular remodeling, as in bone growth. Thus TGF-exposure during articular chondrocytes expansion induces a phenotype switch to hypertrophy, an undesirable effect for cells possibly intended for

  17. Effects of Macromolecular Crowding on Human Adipose Stem Cell Culture in Fetal Bovine Serum, Human Serum, and Defined Xeno-Free/Serum-Free Conditions

    PubMed Central

    Lee, Michelle Hui Ching; Mäkinen, Laura; Ang, Xiu Min; Mannerström, Bettina; Raghunath, Michael; Miettinen, Susanna

    2017-01-01

    Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation. PMID:28465691

  18. Early Prognostic Value of Monitoring Serum Free Light Chain in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation.

    PubMed

    Özkurt, Zübeyde Nur; Sucak, Gülsan Türköz; Akı, Şahika Zeynep; Yağcı, Münci; Haznedar, Rauf

    2017-03-16

    We hypothesized the levels of free light chains obtained before and after autologous stem cell transplantation can be useful in predicting transplantation outcome. We analyzed 70 multiple myeloma patients. Abnormal free light chain ratios before stem cell transplantation were found to be associated early progression, although without any impact on overall survival. At day +30, the normalization of levels of involved free light chain related with early progression. According to these results almost one-third reduction of free light chain levels can predict favorable prognosis after autologous stem cell transplantation.

  19. Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2016-11-24

    The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

  20. Decreased tumorigenicity correlates with expression of altered cell surface carbohydrates in Lec9 CHO cells.

    PubMed Central

    Ripka, J; Shin, S; Stanley, P

    1986-01-01

    To investigate a role for surface carbohydrates in cellular malignancy, 15 different glycosylation-defective CHO cell mutants were examined for their tumorigenic and metastatic capacities after subcutaneous injection into nude mice. Most of the glycosylation mutants displayed similar or slightly decreased tumorigenicity compared with parental CHO cells. Neither parental CHO cells nor any of the mutants were observed to metastasize. However, independent isolates of one mutant type, Lec9, showed a dramatic reduction in tumor formation. The altered carbohydrates expressed at the surface of Lec9 cells appeared to be responsible for their loss of tumorigenicity, because revertants for lectin resistance were able to form tumors, and a double mutant (Lec9.Lec1) that expressed a Lec1 glycosylation phenotype also formed tumors. Finally, Lec9 cells were able to form tumors in gamma-irradiated nude mice, suggesting that recognition by an irradiation-sensitive host cell(s) was responsible for their reduced tumorigenicity in untreated nude mice. PMID:3785164

  1. Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells.

    PubMed

    Freimark, Denise; Sehl, Constanze; Weber, Christian; Hudel, Klaus; Czermak, Peter; Hofmann, Nicola; Spindler, Ralf; Glasmacher, Birgit

    2011-10-01

    Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me(2)SO), is toxic at high concentrations at temperatures >4°C and has harmful effects on the biological functionality of stem cell as well as on treated patients. Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me(2)SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from -16 to -25°C. The CPAs, beside Me(2)SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.

  2. Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

    PubMed Central

    Mignaqui, Ana Clara; Ruiz, Vanesa; Perret, Sylvie; St-Laurent, Gilles; Singh Chahal, Parminder; Transfiguracion, Julia; Sammarruco, Ayelén; Gnazzo, Victoria; Durocher, Yves; Wigdorovitz, Andrés

    2013-01-01

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV. PMID:23977353

  3. An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery.

    PubMed

    Sailer, Martin H M; Sarvepalli, Durga; Brégère, Catherine; Fisch, Urs; Guentchev, Marin; Weller, Michael; Guzman, Raphael; Bettler, Bernhard; Ghosh, Arkasubhra; Hutter, Gregor

    2016-08-23

    Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGFβ-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGFβ-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGFβ-interaction. The results point to a network of FGF-, BMP- and TGFβ-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

  4. Morphological Changes in CHO and VERO Cells Treated with T-2 Mycotoxin. Correlation with Inhibition of Protein Synthesis

    DTIC Science & Technology

    1984-08-21

    Changes in CHO and VERO Cells Treated with T-2 Mycotoxin . Correlation with Publication Inhibition of Protein Synthesis 6. PERFORMING ORG. REPORT...Chinese hamster ovary (CHO) and African green monkey kidney (VERO) cells to T-2 mycotoxin resulted in several morphological changes which appeared to be...Data Entered) . . . .. 0 Morphological Changes in CHO and VERO Cells Treated with T-2 • Mycotoxin . Correlation with Inhibition of Protein Synthesis

  5. LDH-C can be differentially expressed during fermentation of CHO cells.

    PubMed

    Szperalski, Berthold; Jung, Christine; Shao, Zhixin; Kantardjieff, Anne; Hu, Wei-Shou

    2011-01-01

    Expression of CHO mRNA was measured with special microarrays from the Consortium for Chinese Hamster Ovary (CHO) Cell Genomics led by Prof. Wei-Shou Hu of the University of Minnesota and Prof. Miranda Yap of the Bioprocess Technology Institute of A*STAR, Singapore (http://hugroup.cems.umn.edu/CHO/cho_index.html). Cultivation experiments were performed in small scale 2L stirred tank bioreactors. During fermentation a temperature shift of -3°C was performed. This was accompanied by a reduction of the cell specific lactate production rate. The analysis of transcriptome samples before and after the temperature shift with microarrays showed several changes in the expression of available gene markers. LDH-C expression raised about 2 fold after temperature shift. LDH-A did not change. As LDH-C is known to be a specialized isoenzyme in sperm cells for consuming lactate in a lactate containing milieu, LDH-C could be proposed as a target for genetic engineering, facilitating lactate consumption in the late phase of high cell density cultures and prolonging longevity of CHO production cultures by reducing lactate and base accumulation.

  6. Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

    PubMed

    Scher, W; Eto, Y; Ejima, D; Den, T; Svet-Moldavsky, I A

    1990-12-10

    Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.

  7. Production of the Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line in stirred reactor and airlift reactor.

    PubMed

    Micheloud, Gabriela A; Gioria, Verónica V; Eberhardt, Ignacio; Visnovsky, Gabriel; Claus, Juan D

    2011-12-01

    The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), is one of the main plagues for soybean crops. Velvetbean caterpillar larvae are susceptible to be infected by occlusion bodies of the baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a biological insecticide. The insect cell line saUFL-AG-286 produces very high yields of occlusion bodies of AgMNPV in suspension cultures done in the low-cost serum-free medium UNL-10 in shake-flasks. However, its ability to adapt to conditions of industrial production in bioreactors was unknown. The aim of this study was to characterize the growth of saUFL-AG-286 cell cultures in UNL-10 medium, as well as its capability to replicate AgMNPV in two different bio-reactors at laboratory scale. The cell line was able to adapt to conditions that can be used at industrial scale, both in an airlift reactor and a stirred reactor, although the former was better than the last to support the cell growth. The infection with AgMNPV in the airlift reactor produced a high yield of occlusion bodies, with very low production of budded virus, the progeny used as inoculums. On the other hand, infection in the stirred reactor yielded high titers of budded virus. These results suggest that a feasible strategy for scaling-up the production of AgMNPV might involve the use of airlift reactors for the scaling-up of cell suspension cultures and the final production of occlusion bodies, while the scaling-up of the viral inoculums being carried out under conditions as those existing in stirred reactors. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Cholesterol Depletion Facilitates Recovery from Hypotonic Cell Swelling in CHO Cells

    PubMed Central

    Kowalsky, Gregory B.; Beam, Derek; Oh, Myung J.; Sachs, Frederick; Hua, Susan Z.; Levitan, Irena

    2011-01-01

    The maintenance of cell volume homeostasis is critical for preventing pathological cell swelling that may lead to severe cellular dysfunction or cell death. Our earlier studies have shown that volume-regulated anion channels that play a major role in the regulation of cell volume are facilitated by a decrease in cellular cholesterol suggesting that cholesterol depletion should also facilitate regulatory volume decrease (RVD), the ability of cells to recover from hypotonic swelling. In this study, we test this hypothesis using a novel methodology developed to measure changes in cell volume using a microfluidics chamber. Our data show that cholesterol depletion of Chinese Hamster Ovary (CHO) significantly facilitates the recovery process, as is apparent from a faster onset of the RVD (162±10 s. vs. 114±5 s. in control and cholesterol depleted cells respectively) and a higher degree of volume recovery after 10 min of the hypotonic challenge (41%±6% vs. 65%±6% in control and cholesterol depleted cells respectively). In contrast, enriching cells with cholesterol had no effect on the RVD process. We also show here that similarly to our previous observations in endothelial cells, cholesterol depletion significantly increases the stiffness of CHO cells suggesting that facilitation of RVD may be associated with cell stiffening. Furthermore, we also show that increasing cell stiffness by stabilizing F-actin with jasplakinolide also facilitates RVD development. We propose that cell stiffening enhances cell mechano-sensitivity, which in turn facilitates the RVD process. PMID:22179012

  9. Culturing in serum-free culture medium on collagen type-I-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell.

    PubMed

    Arab-Bafrani, Zahra; Shahbazi-Gahrouei, Daryoush; Abbasian, Mehdi; Saberi, Alihossein; Fesharaki, Mehrafarin; Hejazi, Seyed Hossein; Manshaee, Samira

    2016-01-01

    A sub-population of tumor cells termed cancer stem cells (CSCs) has an important role in tumor initiation, progression, and recurrence. Selecting a suitable procedure for isolation and enrichment of CSCs is the biggest challenge in the study of CSCs. In the present study, the role of the combination of stem cell culture medium and collagen type-I was evaluated for successful isolation and enrichment of HT-29 CSCs. HT-29 cells were cultured in serum-containing medium (parental culture medium: Medium + 10% fetal bovine serum) and serum-free medium (stem cell culture medium); both on collagen-coated plates. Spheres forming ability and CD133 expression, as a potential marker of colorectal CSCs, were evaluated in two culture mediums. The results show spheroids usually give rise completely within 15 days in the stem cell culture medium on the collagen-coated plate. CD133 expression in spheroid cells (84%) is extensively higher than in parental cells (25%). Moreover, relative to parental cells, spheroid cells were more radioresistance. Finding of this study suggested that CSCs derived from colon cancer cell line (HT-29) can be propagated and form colonospheres in serum-free culture medium on collagen type-I. According to maintenance of their original phenotype in these conditions, it seems serum-free culture medium on collagen type-I is a suitable way to drug screening of HT-29 CSCs.

  10. A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays

    PubMed Central

    Patel, Asha K.; Celiz, Adam D.; Rajamohan, Divya; Anderson, Daniel G.; Langer, Robert; Davies, Martyn C.

    2016-01-01

    Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 µM in cardiomyocytes cultured on the co-polymer compared to 0.5 µM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials. PMID:26005764

  11. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

    PubMed

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2016-08-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.

  12. A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays.

    PubMed

    Patel, Asha K; Celiz, Adam D; Rajamohan, Divya; Anderson, Daniel G; Langer, Robert; Davies, Martyn C; Alexander, Morgan R; Denning, Chris

    2015-08-01

    Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 μM in cardiomyocytes cultured on the co-polymer compared to 0.5 μM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Selection of High-Producing Clones Using FACS for CHO Cell Line Development.

    PubMed

    Gallagher, Clair; Kelly, Paul S

    2017-01-01

    Cell line development aims to generate and select clones with desirable characteristics. One of the most important parameters for biopharmaceutical cell selection is cell-specific productivity (Qp) or the quantity of product produced per cell per day. Fluorescence-activated cell sorting (FACS) is a powerful, high-throughput technique that facilitates multiparametric characterization and isolation of individual cell clones from heterogeneous populations. Here, we describe a FACS-based method for section of high-producing CHO cell clones.

  14. Computational modelling of genome-scale metabolic networks and its application to CHO cell cultures.

    PubMed

    Rejc, Živa; Magdevska, Lidija; Tršelič, Tilen; Osolin, Timotej; Vodopivec, Rok; Mraz, Jakob; Pavliha, Eva; Zimic, Nikolaj; Cvitanović, Tanja; Rozman, Damjana; Moškon, Miha; Mraz, Miha

    2017-09-01

    Genome-scale metabolic models (GEMs) have become increasingly important in recent years. Currently, GEMs are the most accurate in silico representation of the genotype-phenotype link. They allow us to study complex networks from the systems perspective. Their application may drastically reduce the amount of experimental and clinical work, improve diagnostic tools and increase our understanding of complex biological phenomena. GEMs have also demonstrated high potential for the optimisation of bio-based production of recombinant proteins. Herein, we review the basic concepts, methods, resources and software tools used for the reconstruction and application of GEMs. We overview the evolution of the modelling efforts devoted to the metabolism of Chinese Hamster Ovary (CHO) cells. We present a case study on CHO cell metabolism under different amino acid depletions. This leads us to the identification of the most influential as well as essential amino acids in selected CHO cell lines. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Detachment factors for enhanced carrier to carrier transfer of CHO cell lines on macroporous microcarriers.

    PubMed

    Landauer, K; Dürrschmid, M; Klug, H; Wiederkum, S; Blüml, G; Doblhoff-Dier, O

    2002-05-01

    In this publication different detachment factors were tested for enhancing carrier to carrier transfer for scale-up of macroporous microcarrier based bioprocesses. Two Chinese hamster ovary cell lines, CHO-K1 and a genetically engineered CHO-K1 derived cell line (CHO-MPS), producing recombinant human Arylsulfatase B, were examined. The cells were grown on Cytoline 1microcarriers (Amersham Biosciences, Uppsala, Sweden) in protein-free and chemically defined medium respectively. Fully colonised microcarriers were used at passage ratios of approximately 1:10 for carrier to carrier transfer experiments. To accelerate the colonisation of the non-colonised, freshly added microcarriers the detachment reagents trypsin, papain, Accutasetrade mark (PAA, Linz, Austria), heparin and dextransulphate were used. Both cell lines showed good results with trypsin, Accutase and dextransulphate (Amersham Biosciences, Uppsala, Sweden), while papain failed to enhance carrier to carrier transfer in comparison to the non-treated reference. The maximum growth rate of cells on microcarriers with 2% dextransulphate in the medium was 0.25 +/- 0.02d(-1) and 0.27 +/- 0.03d(-1) for the CHO-MPS and CHO-K1, respectively. TheCHO-K1 grew best after detachment with trypsin (mu = 0.36 +/- 0.03d(-1)). This indicates, that one of the key parameters for carrier to carrier transfer is the uniform distribution of cells on the individual carriers during the initial phase. When this distribution can be improved, growth rate increases, resulting in a faster and more stable process.

  16. Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

    PubMed Central

    Liu, Chia-Chyi; Guo, Meng-Shin; Lin, Fion Hsiao-Yu; Hsiao, Kuang-Nan; Chang, Kate Hsuen-Wen; Chou, Ai-Hsiang; Wang, Yu-Chao; Chen, Yu-Ching; Yang, Chung-Shi; Chong, Pele Choi-Sing

    2011-01-01

    Background Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification. PMID:21603631

  17. A naturally occurring α(s1)-casein-derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum-free conditions.

    PubMed

    Shimizu, T; Ganzorig, K; Miyamoto, A; Ishii, T; Urashima, T; Fukuda, K

    2014-03-01

    Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra-filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α(s1)-CN (f16-23) (RPKHPIKH), α(s1)-CN (f16-24) (RPKHPIKHQ), α(s1)-CN (f17-25) (PKHPIKHQG), α(s1)-CN (f46-52) (VFGKEKV), α(s1)-CN (f94-105) (HIQKEDVPSER), and β-CN (f121-128) (HKEMPFPK), were identified. One of the major peptides, the N-terminal fragment of αs1 -casein, varied structurally during early lactation: α(s1)-CN (f17-25) (PKHPIKHQG) and α(s1)-CN (f16-23) (RPKHPIKH)/α(s1)-CN (f16-24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α(s1)-CN (f16-23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum-free conditions in a dose-dependent manner, in consequence of caspase-3 and caspase-9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti-apoptotic agent in cell culture medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  18. Expression and proteolytic processing of mammalian purple acid phosphatase in CHO-K1 cells.

    PubMed

    Wang, Yunling; Andersson, Göran

    2007-05-01

    Rat recombinant purple acid phosphatase (PAP) stably expressed in fibroblast-like CHO-K1 cells was purified and characterized with respect to post-translational modifications such as N-glycosylation and proteolytic processing in order to elucidate subcellular and molecular pathways for proteolytic activation. In these cells, proteolytically processed PAP was more abundant than the monomeric form. PAP-transfected CHO-K1 cells were expressing active cathepsin K intracellularly, which was partially co-localized with PAP. However, neither cathepsin K nor trypsin digestion of the purified monomeric PAP in vitro did result in a two-subunit form with kinetic and electrophoretic properties resembling the endogenous cellular two-subunit form. Treatment of PAP-transfected CHO-K1 cells with the cysteine proteinase inhibitor E-64 suggested that only a minor fraction of secreted PAP is processed intracellularly by cysteine proteinases. These data do not support a dominant or critical role for cathepsins or trypsin-like serine proteinases in the proteolytic activation of PAP in CHO-K1 cells, implicating yet unidentified proteinases in the proteolytic processing of both intracellular and secreted PAP in this cell line.

  19. Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression.

    PubMed

    Jeong, Yeon Tae; Choi, One; Lim, Hye Rim; Son, Young Dok; Kim, Hong Jin; Kim, Jung Hoe

    2008-12-01

    Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha2,3- sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of alpha2,3-ST and beta1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When alpha2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha2,3-ST and beta1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha2,3- ST and beta1,4-GT is more effective than the expression of alpha2,3-ST alone. Coexpression of alpha2,3-ST and beta1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha2,3-ST and beta1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

  20. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    PubMed Central

    Lee, Jae Seong; Kallehauge, Thomas Beuchert; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup

    2015-01-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production. PMID:25712033

  1. Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting.

    PubMed

    Vcelar, Sabine; Jadhav, Vaibhav; Melcher, Michael; Auer, Norbert; Hrdina, Astrid; Sagmeister, Rebecca; Heffner, Kelley; Puklowski, Anja; Betenbaugh, Michael; Wenger, Till; Leisch, Friedrich; Baumann, Martina; Borth, Nicole

    2017-09-16

    Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8mM glutamine and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  2. Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

    PubMed Central

    Smith, Annie O.; Bowers, Stephanie L. K.; Stratman, Amber N.; Davis, George E.

    2013-01-01

    We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life. PMID:24391990

  3. Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering.

    PubMed

    Lee, Kyoung Ho; Onitsuka, Masayoshi; Honda, Kohsuke; Ohtake, Hisao; Omasa, Takeshi

    2013-07-01

    The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.

  4. Characterization and expression of proprotein convertases in CHO cells: Efficient proteolytic maturation of human bone morphogenetic protein-7.

    PubMed

    Sathyamurthy, Madhavi; Kim, Che Lin; Bang, You Lim; Kim, Young Sik; Jang, Ju Woong; Lee, Gyun Min

    2015-03-01

    Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

  5. Lessons from the hamster: Cricetulus griseus tissue and CHO cell line proteome comparison.

    PubMed

    Heffner, Kelley M; Hizal, Deniz Baycin; Yerganian, George S; Kumar, Amit; Can, Özge; O'Meally, Robert N; Cole, Robert; Chaerkady, Raghothama; Wu, Herren; Bowen, Michael A; Betenbaugh, Michael J

    2017-09-06

    Chinese hamster ovary cells represent the dominant host for therapeutic recombinant protein production. However, few large-scale datasets have been generated to characterize this host organism and derived CHO cell lines at the proteomics level. Consequently, an extensive label-free quantitative proteomics analysis of two cell lines (CHO-S and CHO DG44) and two Chinese hamster tissues (liver and ovary) was used to identify a total of 11801 unique proteins containing at least two unique peptides. 9359 unique proteins were identified specifically in the cell lines, representing a 56% increase over previous work. Additionally, 6663 unique proteins were identified across liver and ovary tissues providing the first Chinese hamster tissue proteome. Protein expression was more conserved within cell lines during both growth phases than across cell lines, suggesting large genetic differences across cell lines. Overall, both gene ontology and KEGG pathway analysis revealed enrichment of cell cycle activity in cells. In contrast, upregulated molecular functions in tissue include glycosylation and lipid transporter activity. Furthermore, cellular components including Golgi apparatus are upregulated in both tissues. In conclusion, this large-scale proteomics analysis enables us to delineate specific changes between tissues and cells derived from these tissues, which can help explain specific tissue function and the adaptations cells incur for applications in biopharmaceutical productions.

  6. Monoclonal and polyclonal gammopathy measured by serum free light chain and immunofixation subdivide the clinical outcomes of diffuse large B-cell lymphoma according to molecular classification.

    PubMed

    Kim, Yu Ri; Kim, Soo-Jeong; Cheong, June-Won; Kim, Yundeok; Jang, Ji Eun; Lee, Jung Yeon; Min, Yoo Hong; Song, Jae-Woo; Yang, Woo Ick; Kim, Jin Seok

    2014-11-01

    Elevated serum free light chain (FLC) is known to be an adverse prognostic factor for diffuse large B-cell lymphoma (DLBCL). We hypothesized that monoclonal gammopathy (MG; elevated kappa [κ] or lambda [λ] FLC with an abnormal κ/λ ratio or a positive IF [immunofixation]) and polyclonal gammopathy (PG; elevated κ and/or λ FLC with a normal κ/λ ratio and a negative IF) would have different clinical outcome according to the molecular classification of DLBCL. In addition, MG would be a poor prognostic factor in patients with activated B-cell like type of DLBCL. Molecular classification of DLBCL, such as germinal center B-cell (GCB) type and non-GCB type, was performed according to the Hans algorithm. Among 175 newly diagnosed DLBCL patients, 96 (54.9 %) patients had an elevated FLC. MG and PG were observed in 34 and 68 patients, respectively. The 2-year overall survival (OS) and event-free survival (EFS) rates were 79.0 % and 71.6 %, respectively. In multivariate analysis, high-intermediate/high International Prognostic Index score and elevated FLC were significant for the OS (P = 0.002, P = 0.005, respectively) and EFS (P < 0.002, P = 0.010, respectively). MG and PG were also associated with inferior OS (P = 0.002, P = 0.011, respectively) and EFS (P = 0.002, P = 0.013, respectively). Ninety-six patients from a total 133 evaluable patients were classified to the non-GCB type. Patients with PG showed inferior clinical outcome for OS and EFS in patients with the GCB type (P = 0.006, P = 0.035, respectively). MG was a significant poor prognostic factor for OS and EFS in patients with the non-GCB type (P = 0.017, P = 0.004, respectively). MG was a poor prognostic maker in patients with the non-GCB type and PG was a poor prognostic indicator for the GCB type of DLBCL who were treated with R-CHOP.

  7. Effects of laser-modified polystyrene substrate on CHO cell growth and alignment.

    PubMed

    Zhu, Bangshang; Lu, Qinhua; Yin, Jie; Hu, Jun; Wang, Zongguang

    2004-07-15

    Biomaterial surface chemistry and nanoscale topography of biomaterials can significantly influence cell behavior in vitro. Polystyrene (PS) Petri dishes were subjected to Nd:YAG laser irradiation at 266 nm, which resulted in well-defined three-dimensional (3D) periodic nanoscale surface topographies and surface oxidation. The surface changes were analyzed by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a contact-angle goniometer. The samples were then used to investigate the cell behavior of Chinese hamster ovary (CHO) cells. The surface laser modification affected the CHO cell adhesion and alignment, and caused morphological changes in comparison with unmodified PS. The results obtained from the cell-behavior studies revealed that nanoscale hydrophilic surface topography cues affected the adhesion, extension, alignment, and morphology of cells. Copyright 2004 Wiley Periodicals, Inc.

  8. In Vitro Chromosome Aberrations Study in Chinese Hamster Ovary (CHO) Cells

    DTIC Science & Technology

    2016-06-07

    Vitro Chromosome Aberrations Study in CHO Cells Duplicate cultures were evaluated for the percentage of first, second, and third plus subsequent...types of aberrations found are presented for each treatment group. The percentage of structurally damaged cells in the total population of cells...Analysis: For each metaphase evaluated, the type and number of aberrations were recorded. The mitotic index (1v1I), percentage of polyploidy among

  9. Modeling shear-induced CHO cell damage in a rotary positive displacement pump.

    PubMed

    Kamaraju, Hari; Wetzel, Kenneth; Kelly, William J

    2010-01-01

    Rotary lobe pumps are commonly used in the biotechnology industry for a variety of purposes. Shear damage to animal cells within the rotary lobe pump can adversely affect the product yield or purity during, for example, cell concentration via cross-flow filtration. In this research, CHO cells grown in 20-L bioreactors were fed to a rotary lobe pump in both single pass and recycle experiments were conducted at different RPMs and "slip" conditions. The results indicate that the slip flow rate more severely impacts the viability of the CHO cells than the pump RPM. A novel mathematical modeling approach is presented that predicts shear rates in all of the positive displacement pump's slip regions, and then predicts cell death vs. operating conditions. This model accounts for the complex flow situation that results from changes to RPM, backpressure and pump geometry (i.e., clearances).

  10. In what time scale proton transfer takes place in a live CHO cell?

    NASA Astrophysics Data System (ADS)

    Mojumdar, Supratik Sen; Chowdhury, Rajdeep; Mandal, Amit Kumar; Bhattacharyya, Kankan

    2013-06-01

    Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) in a live Chinese hamster ovary (CHO) cell is studied by time resolved confocal microscopy. The cytoplasm region of the cell is stained by a photoacid, HPTS (HA). The time constant of initial proton transfer (τPT) in the cell is found to be ˜10 times longer than that in bulk water, while the time constants of recombination (τrec) and dissociation (τdiss) in the cell are ˜3 times and ˜2 times longer, respectively. The slower rate of proton transfer (˜10 times) inside the CHO cell compared to that in bulk water is ascribed to slower solvation dynamics, lower availability of free water molecules, and disruption of hydrogen-bond network inside the cell. Translational and rotational diffusion of HPTS inside a single CHO cell have been investigated by fluorescence correlation spectroscopy (FCS) and picosecond anisotropy measurement, respectively. Both the translational and rotational diffusion slow down inside the live cell. FCS studies indicate that HPTS remains tightly bound to a macromolecule inside the cell.

  11. CHO cells in biotechnology for production of recombinant proteins: current state and further potential.

    PubMed

    Kim, Jee Yon; Kim, Yeon-Gu; Lee, Gyun Min

    2012-02-01

    Recombinant Chinese hamster ovary cells (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Recent advances in cell culture technology for rCHO cells have achieved significant improvement in protein production leading to titer of more than 10 g/L to meet the huge demand from market needs. This achievement is associated with progression in the establishment of high and stable producer and the optimization of culture process including media development. In this review article, we focus on current strategies and achievements in cell line development, mainly in vector engineering and cell engineering, for high and stable protein production in rCHO cells. The approaches that manipulate various DNA elements for gene targeting by site-specific integration and cis-acting elements to augment and stabilize gene expression are reviewed here. The genetic modulation strategy by "direct" cell engineering with growth-promoting and/or productivity-enhancing factors and omics-based approaches involved in transcriptomics, proteomics, and metabolomics to pursue cell engineering are also presented.

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

    PubMed

    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.

  13. Quantitative analysis of the supernatant from host and transfected CHO cells using iTRAQ 8-plex technique.

    PubMed

    Zhu, Guijie; Sun, Liangliang; Albanetti, Thomas; Linkous, Travis; Larkin, Christopher; Schoner, Ronald; McGivney, James B; Dovichi, Norman J

    2016-10-01

    We employed UPLC-MS/MS with iTRAQ 8-plex labeling to quantitatively analyze the supernatant produced by two Chinese hamster ovary (CHO) cell lines (CHO K1SV and CHO CAT-S). In each case, the supernatant from the host and three transfected clones were analyzed at days 5, 7, and 10 of culture. A total of eight iTRAQ 8-plex experiments were performed. For each cell line, the overlap of supernatant protein identifications between transfected clones is over 60%. Over 70% of the supernatant proteins in the CHO K1SV host cell line are present in the CHO CAT-S cell line. For the CHO K1SV cell line, the overlap in supernatant protein identifications between the host cell line and the transfected clones is >59%. For the CHO CAT-S cell line, the overlap between supernatant protein identifications for the transfected clone and host cell is >45%. These differences in the supernatant protein identifications between transfected clones in each cell line and between the two host cell lines are not significant. We used cluster analysis to characterize the change in supernatant protein expression as a function of cell culture time. Roughly <60% of the supernatant proteins show significant change across the three time points (ratio >1.3 or <0.7). We also used cluster analysis to compare changes in supernatant protein expression between the host and three transfected clones at each time point. Greater than 65% of the common proteins in the CHO K1SV cell line supernatant and over 54% in the CHO CAT-S cell line supernatant show no significant expression difference between host and the three transfected clones. Data are available via ProteomeXchange with identifier PXD003462. Biotechnol. Bioeng. 2016;113: 2140-2148. © 2016 Wiley Periodicals, Inc.

  14. Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

    PubMed

    Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

    2014-03-01

    Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products.

  15. Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures.

    PubMed

    Toussaint, Cécile; Henry, Olivier; Durocher, Yves

    2016-01-10

    Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity. Copyright © 2015. Published by Elsevier B.V.

  16. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    NASA Astrophysics Data System (ADS)

    Awasthi, Kumud Kant; Awasthi, Anjali; Kumar, Narender; Roy, Partha; Awasthi, Kamlendra; John, P. J.

    2013-09-01

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 ± 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC50) for CHO cells is 68.0 ± 2.65 μg/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 μg/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  17. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    PubMed

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

  18. Regulatory mutations in CHO cells induce expression of the mouse embryonic antigen SSEA-1.

    PubMed

    Campbell, C; Stanley, P

    1983-11-01

    Two rare and dominant mutants of Chinese hamster ovary (CHO) cells, LEC11 and LEC12, express the mouse embryonic antigen SSEA-1. Parental CHO cells and the revertants, LEC11.R9 and LEC12.R10, do not express this antigen as detected by a sensitive radioimmunoassay with a monoclonal antibody to SSEA-1. The presence of the SSEA-1 determinant correlates with the apparent de novo expression of specific N-acetylglucosaminide alpha(1,3)fucosyltransferase activities not detected in parental or revertant cell extracts. Several differences in the enzymes substrate specificities and their products have been identified. The combined data suggest that LEC11 and LEC12 mutants result from regulatory mutations affecting different fucosyltransferase genes.

  19. Cog2 Null Mutant CHO Cells Show Defective Sphingomyelin Synthesis*

    PubMed Central

    Spessott, Waldo; Uliana, Andrea; Maccioni, Hugo J. F.

    2010-01-01

    The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C6-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II. PMID:21047787

  20. Serum-free media for articular chondrocytes in vitro expansion.

    PubMed

    Shao, Xin-xin; Duncan, Neil A; Lin, Lin; Fu, Xin; Zhang, Ji-ying; Yu, Chang-long

    2013-07-01

    In vitro chondrocyte expansion is a major challenge in cell-based therapy for human articular cartilage repair. Classical culture conditions usually use animal serum as a medium supplement, which raises a number of undesirable questions. In the present study, two kinds of defined, serum-free media were developed to expand chondrocytes in monolayer culture for the purpose of cartilage tissue engineering. Bovine chondrocytes were expanded in serum-free media supplemented with fibroblast growth factor-2 and platelet-derived growth factor or fibroblast growth factor-2 and insulin-like growth factor. Expansion culture in a conventional 10% fetal bovine serum (FBS) medium served as control. Fibronectin coating was used to help cell adhesion in serum-free medium. Next, in vitro three-dimensional pellet culture was used to evaluate the chondrocyte capacity. Cell pellets were expanded in different media to re-express the differentiated phenotype (re-differentiation) and to form cartilaginous tissue. The pellets were assessed by glycosaminoglycans contents, collagen II, collagen I and collagen X immunohistological staining. Chondrocytes cultured in serum-free media showed no proliferation difference than cells grown with 10% FBS medium. In addition, chondrocytes expanded in both serum-free media expressed more differentiated phenotypes at the end of monolayer culture, as indicated by higher gene expression ratios of collagen type II to collagen type I. Pellets derived from chondrocytes cultured in both serum-free media displayed comparable chondrogenic capacities to pellets from cells expanded in 10% FBS medium. These findings provide alternative culture approaches for chondrocytes in vitro expansion, which may benefit the clinical use of autologous chondrocytes implantation.

  1. Expression of GPI anchored human recombinant erythropoietin in CHO cells is devoid of glycosylation heterogeneity.

    PubMed

    Singh, Pankaj Kumar; Devasahayam, Mercy; Devi, Sobita

    2015-04-01

    Erythropoietin is a glycohormone involved in the regulation of the blood cell levels. It is a 166 amino acid protein having 3 N-glycosylation and one O-linked glycosylation sites, and is used to treat anaemia related illness. Though human recombinant erythropoietin (rEPO) is produced in CHO cells, the loss in quality control is 80% due to incomplete glycosylation of the rEPO with low levels of fully glycosylated active rEPO. Here, we describe the expression from CHO cells of fully glycosylated human rEPO when expressed as a GPI anchored molecule (rEPO-g). The results demonstrated the production of a homogenous completely glycosylated human rEPO-g as a 42 kD band without any low molecular weight glycoform variants as shown by affinity chromatography followed by SDS-PAGE and anti-human EPO specific western blot. The western blot using specific monoclonal antibody is the available biochemical technique to prove the presence of homogeneity in the expressed recombinant protein. The GPI anchor can be removed during the purification process to yield a therapeutically relevant recombinant erythropoietin molecule cells with a higher in vivo biological activity due to its high molecular weight of 40 kD. This is possibly the first report on the production of a homogenous and completely glycosylated human rEPO from CHO cells for efficient therapy.

  2. Production of a Chikungunya Vaccine Using a CHO Cell and Attenuated Viral-Based Platform Technology.

    PubMed

    Eldi, Preethi; Cooper, Tamara H; Liu, Liang; Prow, Natalie A; Diener, Kerrilyn R; Howley, Paul M; Suhrbier, Andreas; Hayball, John D

    2017-10-04

    Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Poly-γ-glutamic acid enhances the quality of recombant erythropoietin produced by CHO cells

    PubMed Central

    Kim, Tae Gon; Cho, Young Chol; Chun, Bok-Hwan; Park, Sung Hyo; Lee, Hoi-Seon; Chung, Namhyun

    2014-01-01

    The effect of poly-γ-glutamic acid (γPGA), which is produced by Bacillus sp., on the production of recombinant erythropoietin (rEPO) by Chinese hamster ovary (CHO) cells in suspension culture was evaluated. The growth, viability, and productivity of recombinant CHO cells were investigated in a chemically defined medium with 50 and 500 kD γPGAs at 0.075% or with Pluronic F68. Cell growth with the two γPGAs was lower than that with Pluronic F68 but significantly higher than that without any additive (control). The effect of additives on rEPO productivity was 50 kDa γPGA > 500 kDa γPGA > Pluronic F68 > control. Using EPO-dependent F-36E cells, we found that the effect of additives on rEPO quality was 500 kDa γPGA > 50 kDa γPGA > control > Pluronic F68. γPGA has an enhancement effect on the quality of rEPO produced by CHO cells. PMID:26019520

  4. Poly-γ-glutamic acid enhances the quality of recombant erythropoietin produced by CHO cells.

    PubMed

    Kim, Tae Gon; Cho, Young Chol; Chun, Bok-Hwan; Park, Sung Hyo; Lee, Hoi-Seon; Chung, Namhyun

    2014-03-04

    The effect of poly-γ-glutamic acid (γPGA), which is produced by Bacillus sp., on the production of recombinant erythropoietin (rEPO) by Chinese hamster ovary (CHO) cells in suspension culture was evaluated. The growth, viability, and productivity of recombinant CHO cells were investigated in a chemically defined medium with 50 and 500 kD γPGAs at 0.075% or with Pluronic F68. Cell growth with the two γPGAs was lower than that with Pluronic F68 but significantly higher than that without any additive (control). The effect of additives on rEPO productivity was 50 kDa γPGA > 500 kDa γPGA > Pluronic F68 > control. Using EPO-dependent F-36E cells, we found that the effect of additives on rEPO quality was 500 kDa γPGA > 50 kDa γPGA > control > Pluronic F68. γPGA has an enhancement effect on the quality of rEPO produced by CHO cells.

  5. Serum-Free Culture of the Suspension Cell Line QB-Tn9-4s of the Cabbage Looper, Trichoplusia ni, is Highly Productive for Virus Replication and Recombinant Protein Expression

    PubMed Central

    Zheng, Gui-Ling; Zhou, Hong-Xu; Li, Chang-You

    2014-01-01

    Serum-free cultures of insect cells play an important role in the fields of protein engineering, medicine, and biology. In this paper, the suspension cell line QB-Tn9-4s of Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) was successfully adapted to serum-free Sf-900 III medium and passaged for 52 generations. The adapted QB-Tn9-4s cells grew faster. Their population doubling time shortened from 27.4 hr in serum-containing medium to 24.1 hr, and their maximal density increased by 1.83-fold, reaching 3.50 × 106 cells/mL in serum-free culture in T-flasks. The cells readily adapted to spinner culture, with maximum cell density of 4.40 × 106 cells/mL in a spinner flask. Although the infection rate of Autographa californica multiple nucleopolyhedrovirus and production of occlusion bodies (OBs) of the adapted QB-Tn9-4s cells were 91.0% and 85.4 OBs/cell, respectively, similar to those of QB-Tn9-4s cells cultured in serum-containing medium and control BTI-Tn5B1-4 cells, their budded virus titer was 4.97 × 107 TCID50/mL, significantly higher than those of the latter two. In addition, the expression levels of β-galactosidase at six days post-infection and secreted alkaline phosphatase at seven days postinfection in the adapted QB-Tn9-4s cells reached 2.98 ± 0.15×104 IU/mL and 3.34 ± 0.13 IU/mL, respectively, significantly higher than those of QB-Tn9-4s and control BTI-Tn5B1-4 cultured in serum-containing media. The above findings establish a foundation for industrial production of virus and recombinant proteins in QB-Tn9-4s serum-free culture. PMID:25373171

  6. A framework to quantify karyotype variation associated with CHO cell line instability at a single-cell level.

    PubMed

    Baik, Jong Youn; Lee, Kelvin H

    2017-05-01

    Chinese hamster ovary (CHO) cells, the major mammalian host cells for biomanufacturing of therapeutic proteins, have been extensively investigated to enhance productivity and product quality. However, cell line instability resulting in unexpected changes in productivity or product quality continues to be a challenge. Based on previous reports about causes and characteristics of production instability, we hypothesized that chromosomal rearrangements due to genomic instability are associated with production instability and that these events can be characterized. We developed a production instability model using secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) as well as a framework to quantify chromosomal rearrangements by karyotyping. In the absence of methotrexate (MTX), CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes. In contrast, when MTX was re-introduced, the growth rate and SEAP productivity reversed to the initial values, demonstrating the reversibility of production instability in CHO-SEAP cells. Fluorescence in situ hybridization analysis identified that the SEAP genes were incorporated in the chromosomal rearrangement (insertion) part of the der(Z9) chromosome. Karyotype analysis indicated that the insertion ratio of the der(Z9) chromosome decreased in the CHO-SEAP cells grown without MTX, demonstrating a correlation between chromosomal rearrangement and production instability. Our results support a mechanism for production instability, wherein a randomly generated chromosomal rearrangement (or genotype) results in cells with a growth advantage that is also associated with non (or low)-producing traits. As a result, the non-producing cells grow faster and thereby outgrow the producing population. Biotechnol. Bioeng. 2017;114: 1045-1053. © 2016 Wiley Periodicals, Inc.

  7. Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

    PubMed

    Lim, U Ming; Yap, Miranda Gek Sim; Lim, Yoon Pin; Goh, Lin-Tang; Ng, Say Kong

    2013-07-05

    Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.

  8. CMV promoter mutants with a reduced propensity to productivity loss in CHO cells

    PubMed Central

    Moritz, Benjamin; Becker, Peter B.; Göpfert, Ulrich

    2015-01-01

    The major immediate-early promoter and enhancer of the human cytomegalovirus (hCMV-MIE) is one of the most potent DNA elements driving recombinant gene expression in mammalian cells. Therefore, it is widely employed not only in research but also in large-scale industrial applications, e.g. for the production of therapeutic antibodies in Chinese hamster ovary cells (CHO). As we have reported previously, multi-site methylation of hCMV-MIE is linked to productivity loss in permanently transfected CHO cells lines. In particular, the cytosine located 179 bp upstream of the transcription start site (C-179) is frequently methylated. Therefore, our objective was to study whether mutation of C-179 and other cytosines within hCMV-MIE might lessen the instability of transgene expression. We discovered that the single mutation of C-179 to G can significantly stabilise the production of recombinant protein under control of hCMV-MIE in permanently transfected CHO cells. PMID:26581326

  9. Elucidating the role of copper in CHO cell energy metabolism using (13)C metabolic flux analysis.

    PubMed

    Nargund, Shilpa; Qiu, Jinshu; Goudar, Chetan T

    2015-01-01

    (13)C-metabolic flux analysis was used to understand copper deficiency-related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein-producing CHO cells. Stationary-phase labeling experiments with U-(13)C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed-batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC-MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%-79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%-23% and 74%, respectively) compared with the Cu-containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper-deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation.

  10. Endogenous TRPM4-like channel in Chinese hamster ovary (CHO) cells

    SciTech Connect

    Yarishkin, Oleg V.; Hwang, Eun-Mi; Park, Jae-Yong; Kang, Dawon; Han, Jaehee; Hong, Seong-Geun

    2008-05-02

    Chinese hamster ovary (CHO) cells used in many transfection studies have been found to endogenously express channels permeable to monovalent cations, but not to divalent cations. In the presence of intracellular Ca{sup 2+}, 23-pS channel with a linear current-voltage (I-V) relationship could be frequently observed in inside-out patches but not in cell-attached patches. The open probability was voltage-dependent, which is higher at positive potentials. The channel was dose-dependently activated by relatively high level of Ca{sup 2+} (EC{sub 50} = 1.04 {+-} 0.08 mM), and sensitively inhibited by 100 {mu}M ATP, ADP, AMP, and 1 mM spermine. However, ruthenium red (2 {mu}M) had no effect. Reverse transcript polymerase chain reaction (RT-PCR) supported the presence of mRNA encoding TRPM4b channel protein. Western blot assay finally confirmed the presence of this channel protein in membrane fraction of CHO cells. These results provide evidence that CHO cells express an endogenous TRPM4b-like channel, and thereby can be used as a tool to study de novo regulation/modulation of TRPM4 channel.

  11. Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

    NASA Astrophysics Data System (ADS)

    Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

    2014-03-01

    Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to

  12. Comparative Metabolic Analysis of CHO Cell Clones Obtained through Cell Engineering, for IgG Productivity, Growth and Cell Longevity

    PubMed Central

    Wilkens, Camila A.; Gerdtzen, Ziomara P.

    2015-01-01

    Cell engineering has been used to improve animal cells’ central carbon metabolism. Due to the central carbon metabolism’s inefficiency and limiting input of carbons into the TCA cycle, key reactions belonging to these pathways have been targeted to improve cultures’ performance. Previous works have shown the positive effects of overexpressing PYC2, MDH II and fructose transporter. Since each of these modifications was performed in different cell lines and culture conditions, no comparisons between these modifications can be made. In this work we aim at contrasting the effect of each of the modifications by comparing pools of transfected IgG producing CHO cells cultivated in batch cultures. Results of the culture performance of engineered clones indicate that even though all studied clones had a more efficient metabolism, not all of them showed the expected improvement on cell proliferation and/or specific productivity. CHO cells overexpressing PYC2 were able to improve their exponential growth rate but IgG synthesis was decreased, MDH II overexpression lead to a reduction in cell growth and protein production, and cells transfected with the fructose transporter gene were able to increase cell density and reach the same volumetric protein production as parental CHO cells in glucose. We propose that a redox unbalance caused by the new metabolic flux distribution could affect IgG assembly and protein secretion. In addition to reaction dynamics, thermodynamic aspects of metabolism are also discussed to further understand the effect of these modifications over central carbon metabolism. PMID:25768021

  13. [Effect of Intron Orientation on the Expression of Transgene Imposed by MAR Expression Vector in Stably Recombinant CHO Cells].

    PubMed

    Li, Qin; Zhao, Chun-peng; Wang, Xiao-yin; Sun, Qiu-li; Wang, Tian-yun

    2016-03-01

    To determine the effect of intron orientation on the transgene expression level imposed by matrix attachment region (MAR) expression vector. The MAR of β-globin was amplified by PCR, and then cloned into MAR expression vectors. An intron sequence was digested with restriction enzyme, ligated to the MAR expression vector in reverse orientation, and then transfected into Chinese hamster ovary (CHO) cells. The transfected stable cells were screened by G418. The level of chloramphenicol acetyltransferase (CAT) gene expression was analyzed by ELISA method. The transgene expression levels of CHO cells with the two expression vectors with a positive intron or without MAR were higher than that of CHO cells with an expression vector with reverse intron (P < 0.05). MAR did not improve transgene expression with reverse intron presence. Different orientation of intron can affect transgene expression in recombinant CHO cells. The transgene expression level can be increased using positive intron and MAR.

  14. Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

    PubMed

    Chung, Cheng-Yu; Wang, Qiong; Yang, Shuang; Ponce, Sean A; Kirsch, Brian J; Zhang, Hui; Betenbaugh, Michael J

    2017-07-07

    One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications. © 2017 Wiley Periodicals, Inc.

  15. The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker.

    PubMed

    Keen, M J; Hale, C

    1995-01-01

    A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H(*) were adapted to enable them to grow serum-free in the absence of cholesterol and β-cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10(6) cells ml(-1) producing 76.6 mg l(-1) of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.

  16. Analysis of Membrane Topology of Prestin Expressing in CHO Cells

    NASA Astrophysics Data System (ADS)

    Murakoshi, Michio; Kawase, Tomohiro; Kumano, Shun; Wada, Hiroshi

    2011-11-01

    Outer hair cell (OHC) motility is thought to be based on the voltage-dependent conformational changes of the motor protein prestin. However, little is known about its structure and function. In this study, the membrane topology of prestin was investigated by single molecule force spectroscopy using an atomic force microscope (AFM). The C-terminus of prestin was tagged with an Avi-tag and biotinylated. Prestin was then connected with a streptavidin-coated AFM cantilever via biotin-streptavidin binding. The prestin was pulled out from the plasma membrane by retracting the cantilever and force curves were obtained. Obtained force curves suggested the existence of 12 transmembrane domains of prestin.

  17. Production of occlusion bodies of Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line: influence of infection conditions and statistical optimization.

    PubMed

    Micheloud, Gabriela A; Gioria, Verónica V; Pérez, Gustavo; Claus, Juan D

    2009-12-01

    The influence of the conditions of infection on the yield of occlusion bodies (OBs) of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), produced in serum-free suspension cultures of saUFL-AG-286 cells, was investigated by two 2(2) full factorial experiments with centre point. Each experiment tested the effects of the initial cell density and the multiplicity of infection at two levels, in the four possible combinations of levels and conditions, plus a further combination with each condition set at the middle of its extreme levels. The yield of occlusion bodies proved to be sensitive to the modification of infection conditions. Maximum yield as high as 3 x 10(8) OBs mL(-1) was attained provided that the maximum density of viable cells was in the range between 4 and 8 x 10(5) cells mL(-1). The optimum value of the maximum density of viable cells could be reached by the combination of several values of initial cell density and multiplicity of infection. A regression model was established and validated in order to optimize the infection conditions. These results demonstrate the importance of an adequate selection of infection conditions, and they could be useful in the development of a feasible in vitro process to produce the AgMNPV insecticide in a new serum-free medium.

  18. Effects of selenocystine on lead-exposed Chinese hamster ovary (CHO) and PC-12 cells

    SciTech Connect

    Aykin-Burns, Nukhet; Ercal, Nuran . E-mail: nercal@umr.edu

    2006-07-15

    Lead is a pervasive environmental toxin that affects multiple organ systems, including the nervous, renal, reproductive, and hematological systems. Even though it is probably the most studied toxic metal, some of the symptoms of lead toxicity still cannot be explained by known molecular mechanisms. Therefore, lead-induced oxidative stress has recently started to gain attention. This in vitro study confirms the existence of oxidative stress due to lead exposure. Administration of lead acetate (PbA) to cultures of Chinese hamster ovary cells (CHO) had a concentration-dependent inhibitory effect on colony formation and cell proliferation. This inhibition was eliminated by 5 {mu}M selenocystine (SeCys). In order to evaluate the nature of SeCys's effect, we measured glutathione (GSH), its oxidized form glutathione disulfide (GSSG), malondialdehyde (MDA), catalase, and GSH peroxidase (GPx) activities in lead-exposed CHO cells both in the presence and absence of SeCys. Increases in MDA, catalase, and GPx activities were observed in cultures that received only PbA, but supplementation with SeCys returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in SeCys-enhanced media but declined in cultures treated with PbA only. In order to determine whether SeCys also reverses lead-induced neurotoxicity, a neuronal cell line, PC-12 cells, was used. Lead's inhibition on neurite formation was significantly eliminated by SeCys in PC-12 cells. Our results suggest that SeCys can confer protection against lead-induced toxicity in CHO cells and neurotoxicity in PC-12 cells.

  19. Adhesion and migration of CHO cells on micropatterned single layer graphene

    NASA Astrophysics Data System (ADS)

    Keshavan, S.; Oropesa-Nuñez, R.; Diaspro, A.; Canale, C.; Dante, S.

    2017-06-01

    Cell patterning technology on single layer graphene (SLG) is a fairly new field that can find applications in tissue engineering and biomaterial/biosensors development. Recently, we have developed a simple and effective approach for the fabrication of patterned SLG substrates by laser micromachining, and we have successfully applied it for the obtainment of geometrically ordered neural networks. Here, we exploit the same approach to investigate the generalization of the cell response to the surface cues of the fabricated substrates and, contextually, to quantify cell adhesion on the different areas of the patterns. To attain this goal, we tested Chinese hamster ovary (CHO) cells on PDL-coated micropatterned SLG substrates and quantified the adhesion by using single cell force spectroscopy (SCFS). Our results indicate higher cell adhesion on PDL-SLG, and, consequently, an initial CHO cell accumulation on the graphene areas, confirming the neuronal behaviour observed previously; interestingly, at later time point in culture, cell migration was observed towards the adjacent SLG ablated regions, which resulted more favourable for cell proliferation. Therefore, our findings indicate that the mechanism of interaction with the surface cues offered by the micropatterned substrates is strictly cell-type dependent.

  20. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    SciTech Connect

    Lau, Aik Jiang; Chang, Thomas K.H.

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  1. Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

    PubMed Central

    Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

    2017-01-01

    Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

  2. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  3. Host cell protein dynamics in recombinant CHO cells: impacts from harvest to purification and beyond.

    PubMed

    Hogwood, Catherine Em; Bracewell, Daniel G; Smales, C Mark

    2013-01-01

    During the production of recombinant protein products, such as monoclonal antibodies, manufacturers must demonstrate clearance of host cell impurities and contaminants to appropriate levels prior to use in the clinic. These include host cell DNA and RNA, product related contaminants such as aggregates, and importantly host cell proteins (HCPs). Despite the importance of HCP removal, the identity and dynamics of these proteins during cell culture and downstream processing (DSP) are largely unknown. Improvements in technologies such as SELDI-TOF mass spectrometry alongside the gold standard technique of ELISA has allowed semi-quantification of the total HCPs present. However, only recently have techniques been utilized in order to identify those HCPs present and align this with the development of approaches to monitor the dynamics of HCPs during both fermentation and downstream processing. In order to enable knowledge based decisions with regards to improving HCP clearance it is vital to identify potential problematic HCPs on a cell line and product specific basis. Understanding the HCP dynamics will in the future help provide a platform to rationally manipulate and engineer and/or select suitable recombinant CHO cell lines and downstream processing steps to limit problematic HCPs.

  4. Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

    PubMed

    Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

    2017-02-01

    Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h(-1) and 0.044 h(-1) were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h(-1) in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

  5. Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

    PubMed

    Juárez, Mariana; González-De la Rosa, Claudia H; Memún, Elisa; Sigala, Juan-Carlos; Lara, Alvaro R

    2017-03-01

    Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.

  6. The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside.

    PubMed

    Takagi, Yasuhiro; Kikuchi, Takuya; Wada, Ryuta; Omasa, Takeshi

    2017-03-02

    Recently, with the dramatic increase in demand for therapeutic antibodies, Chinese hamster ovary (CHO) cell culture systems have made significant progress in recombinant antibody production. Over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. Medium optimization has been identified as an important key approach for increasing product concentrations. In this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody-expressing CHO cell lines. Furthermore, we investigated the effects of combined addition of deoxyuridine, thymidine, and deoxycytidine. Our results suggest that addition of these pyrimidine nucleosides can increase CHO cell growth, with no significant change in the specific production rate. As a result of the increased cell growth, the antibody concentration was elevated and we were able to achieve more than 9 g/L during 16 days of culture. Similar effects of nucleoside addition were observed in fed-batch cultures of a Fab fragment-expressing CHO cell line, and the final Fab fragment concentration was more than 4 g/L. This nucleoside addition strategy could be a powerful platform for efficient antibody production.

  7. Synchronization of mitochondrial DNA synthesis in Chinese hamster cells (line CHO) deprived of isoleucine.

    PubMed

    Ley, K D; Murphy, M M

    1973-08-01

    Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([(3)H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([(3)H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9-12 h after addition of isoleucine and virtually all [(3)H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G(1) because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

  8. miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

    PubMed

    Fischer, Simon; Paul, Albert Jesuran; Wagner, Andreas; Mathias, Sven; Geiss, Melanie; Schandock, Franziska; Domnowski, Martin; Zimmermann, Jörg; Handrick, René; Hesse, Friedemann; Otte, Kerstin

    2015-10-01

    Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.

  9. Genetically modified CHO cells for studying the genotoxicity of heterocyclic amines from cooked foods

    SciTech Connect

    Thompson, L.H.; Wu, R.W.; Felton, J.S.

    1995-07-01

    We have developed metabolically competent CHO cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with IQ but not with PhIP. This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines.

  10. Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

    SciTech Connect

    Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

    2010-04-23

    Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

  11. Air sparging for prevention of antibody disulfide bond reduction in harvested CHO cell culture fluid.

    PubMed

    Mun, Melissa; Khoo, Stefanie; Do Minh, Aline; Dvornicky, James; Trexler-Schmidt, Melody; Kao, Yung-Hsiang; Laird, Michael W

    2015-04-01

    During the scale-up of several Chinese Hamster Ovary (CHO) cell monoclonal antibody production processes, significant reduction of the antibody interchain disulfide bonds was observed. The reduction was correlated with excessive mechanical cell shear during the harvest operations. These antibody reduction events resulted in failed product specifications and the subsequent loss of the drug substance batches. Several methods were recently developed to prevent antibody reduction, including modifying the cell culture media, using pre- and post-harvest chemical additions to the cell culture fluid (CCF), lowering the pH, and air sparging of the harvested CCF (HCCF). The work described in this paper further explores the option of HCCF air sparging for preventing antibody reduction. Here, a small-scale model was developed using a 3-L bioreactor to mimic the conditions of a manufacturing-scale harvest vessel and was subsequently employed to evaluate several air sparging strategies. In addition, these studies enabled further understanding of the relationships between cell lysis levels, oxygen consumption, and antibody reduction. Finally, the effectiveness of air sparging for several CHO cell lines and the potential impact on product quality were assessed to demonstrate that air sparging is an effective method in preventing antibody reduction.

  12. The influence of ciprofloxacin on hamster ovarian cancer cell line CHO AA8.

    PubMed

    Kloskowski, Tomasz; Olkowska, Joanna; Nazlica, Aybars; Drewa, Tomasz

    2010-01-01

    Abstract: Ciprofloxacin is a chinolone antibiotic, which is used mainly in the treatment of urinary tract infections but also in pulmonary tract, prostate gland, bone and bone marrow infection. Ciprofloxacin is also known for its anticancer in vitro properties. In this study hamster ovarian cancer line CHO AA8 was used for evaluation of cytotoxic properties of ciprofloxacin against neoplastic cells. For this purpose we used different concentrations of ciprofloxacin range from 10 to 1000 microg/mL. Cell viability was counted using trypan blue assay. Ciprofloxacin induced morphological changes and decreased viability in a concentration and time dependent manner within CHO AA8 cells. In low concentrations cytotoxic effect of ciprofloxacin is weak only after 24 h incubation. In the highest concentration of ciprofloxacin, after 24, 48 and 72 h incubation only a very small number of living cells (not exceeding 1%) was observed. No living cells were observed after 96 h of incubation times and ciprofloxacin concentrations of 800 and 1000 micrpg/mL. These promising results deserved future studies on chinolones and ovarian cancer.

  13. Effects of high passage cultivation on CHO cells: a global analysis.

    PubMed

    Beckmann, T F; Krämer, O; Klausing, S; Heinrich, C; Thüte, T; Büntemeyer, H; Hoffrogge, R; Noll, T

    2012-05-01

    Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.

  14. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    PubMed

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

  15. Approaches to maximizing stable expression of alpha 1-antitrypsin in transformed CHO cells.

    PubMed

    Paterson, T; Innes, J; Moore, S

    1994-01-01

    A variety of approaches to maximizing the production of recombinant human alpha 1-antitrypsin (AAT) in Chinese hamster ovary (CHO) cells have been investigated. The highly active and inducible human cytomegalovirus immediate early (IE) promoter/enhancer was used to drive transcription of a recombinant AAT gene in transiently transfected and stably transformed CHO cells. The AAT gene was modified to incorporate highly efficient 3'RNA processing signals from the herpes simplex virus type 2 IE gene 5, and optimal translational initiation signals were created by site-directed mutagenesis. The effect of flanking the recombinant gene with matrix attachment regions was investigated. Combinations of these modifications allowed secretion of up to 44 micrograms AAT/ml per day by cell lines growing in serum-rich medium. This could be increased to up to 100 micrograms AAT/ml per day upon chemical induction of expression by propionate, butyrate or hexamethylene bisacetamide. Cell lines adapted to grow in protein-free medium produced less AAT but still responded to chemical induction to secrete up to 14 micrograms/ml per day of readily purified AAT.

  16. The characterization and quantitation of glycomic changes in CHO cells during a bioreactor campaign.

    PubMed

    Tep, Samnang; Hincapie, Marina; Hancock, William S

    2012-12-01

    Within the biotechnology industry there is a continuous drive to better and more fully understand the biopharmaceutical process in order to achieve better process control. A method to monitor and quantitate glycomic changes that occur in CHO cells during a bioreactor campaign could help to address this. The goal of the method presented here is to provide data that may help to understand the changes in glycosylation that are occurring, within the cell, to proteins other than the expressed biotherapeutic. The method involves the lysing of cells to gain access to intracellular proteins. The expressed biotherapeutic is specifically removed by affinity chromatography, while the remaining proteins are subjected to deglycosylation by treatment with PNGase F. The released glycans are derivatized with isotopic tags, and quantitative analysis by MALDI-TOF MS is performed. The MALDI-TOF MS method allows for the simultaneous analysis of both neutral and sialylated glycans, displays a linear dynamic range over two orders of magnitude for both neutral and sialylated glycans and achieves sub-picomolar sensitivity. This method may yield valuable information that gives further insight into the inner-workings of CHO cells, potentially taking another step towards fully understanding and controlling the biopharmaceutical process.

  17. Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Larson, Jeffery L; Bowden, Daniel M; Frye, Christopher C

    2013-12-01

    Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO

  18. Benchmarking of commercially available CHO cell culture media for antibody production.

    PubMed

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-06-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable

  19. Analytical Criticalities Associated to Different Immunological Methods for Serum Free Light Chain Detection in Plasma Cell Dyscrasias: A Description of Particular Clinical Cases.

    PubMed

    Sabatino, Rocco; Perrone, Antonio; Cuomo, Marco; Liotti, Sandra; Barchiesi, Vittoria; Cantile, Monica; Cavalcanti, Ernesta

    2017-04-12

    Current criteria for differential diagnosis of multiple myeloma (MM), Monoclonal gammopathy of undetermined significance (MGUS), and smoldering multiple myeloma (SMM) are included in the 2003 guidelines by the International Myeloma Working Group (IMWG). An updated version was then published in 2014, highlighting the importance of serum free light chain (sFLC) detection, as well as the κ/λ ratio as excellent indicators of clonality. At present, two commercial assays for sFLC quantification are available: the Freelite™ assay and the N-Latex assay. The first was developed by The Binding Site based on a mixture of polyclonal antibodies directed against a variety of FLC epitopes. It may be run on a wide range of nephelometers, as well as on turbidimeters. The second method was developed by Siemens and runs exclusively on Siemens instruments. It employs a probe mixture of mouse monoclonal antibodies. The aim of our study was to evaluate sFLC measurement and calculated κ/λ ratio in 85 patients with monoclonal gammopathies (MGs) in order to compare methods. We demonstrated that there is only a moderate concordance between the two FLC assays. In particular, in one case, we observed no qualitative alterations of the serum protein pattern, and in the absence of a Freelite™ assay, sFLC measurement would not have been possible to highlight the increase of λ FLC.

  20. CHO cell enlargement oscillates with a temperature-compensated period of 24 min

    NASA Technical Reports Server (NTRS)

    Pogue, R.; Morre, D. M.; Morre, D. J.

    2000-01-01

    The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14 degrees C, 24 degrees C or 34 degrees C) while the rate of cell enlargement increased with temperature over this same range of temperatures.

  1. CHO cell enlargement oscillates with a temperature-compensated period of 24 min

    NASA Technical Reports Server (NTRS)

    Pogue, R.; Morre, D. M.; Morre, D. J.

    2000-01-01

    The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14 degrees C, 24 degrees C or 34 degrees C) while the rate of cell enlargement increased with temperature over this same range of temperatures.

  2. Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin

    PubMed Central

    Baik, Jong Youn; Dahodwala, Hussain; Oduah, Eziafa; Talman, Lee; Gemmill, Trent R.; Gasimli, Leyla; Datta, Payel; Yang, Bo; Li, Guoyun; Zhang, Fuming; Li, Lingyun; Linhardt, Robert J.; Campbell, Andrew M.; Gorfien, Stephen F.; Susan, T. Sharfstein

    2015-01-01

    Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S® cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ~100-fold and the heparin/heparan sulfate yield ~10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ~two-fold increase in integrated viable cell density and 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ~90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug. PMID:26037948

  3. Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time

    PubMed Central

    Feichtinger, Julia; Hernández, Inmaculada; Fischer, Christoph; Hanscho, Michael; Auer, Norbert; Hackl, Matthias; Jadhav, Vaibhav; Baumann, Martina; Krempl, Peter M.; Schmidl, Christian; Farlik, Matthias; Schuster, Michael; Merkel, Angelika; Sommer, Andreas; Heath, Simon; Rico, Daniel; Bock, Christoph; Thallinger, Gerhard G.

    2016-01-01

    ABSTRACT The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA‐methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA‐methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241–2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:27072894

  4. Exploration of BAC versus plasmid expression vectors in recombinant CHO cells.

    PubMed

    Mader, Alexander; Prewein, Bernhard; Zboray, Katalin; Casanova, Emilio; Kunert, Renate

    2013-05-01

    Vector engineering approaches are commonly used to increase recombinant protein production in mammalian cells, and among various concepts, bacterial artificial chromosomes (BAC) have been proposed to serve as open chromatin regions to omit chromosome positional effects. For proof of concept, we developed stable recombinant Chinese hamster ovary (CHO) cell lines using different expression vector systems: the plasmid vectors contained the identical expression cassette as the BAC constructs. Two anti-HIV1 antibody derivates served as model proteins (3D6scFc and 2F5scFc) for generation of four stable recombinant CHO cell lines. The BAC-derived clones showed three to four times higher specific productivity, and therefore, gene copy numbers and transcript level were quantified. The active chromatin region provided with the BAC environment significantly improved transcription evidenced with both model proteins. Specific transcription was approximately six times higher from BAC-based vectors compared to the corresponding plasmid vectors for both single-chain fragment crystallizable (scFc) proteins. Our accurate investigations elucidated also differences between translational activities related to the protein of choice. 3D6scFc expressed specifically three to four times more product than 2F5scFc indicating that the product by itself also contributes to enhanced productivity. This study indicated comparable increase of transcription level for both scFc proteins when using the BAC system, but translation, maturation, and secretion of individual proteins seem to be protein specific.

  5. Overexpression of OSBP-related protein 2 (ORP2) in CHO cells induces alterations of phospholipid species composition.

    PubMed

    Käkelä, Reijo; Tanhuanpää, Kimmo; Laitinen, Saara; Somerharju, Pentti; Olkkonen, Vesa M

    2005-10-01

    We have previously shown that overexpression of human OSBP-related protein 2 (ORP2) in Chinese hamster ovary (CHO) cells results in increased efflux and reduced esterification of cholesterol. The ORP2-expressing cells also have a reduced level of triacylglycerols. We investigated the effects of ORP2 expression on the phospholipid (PL) molecular species and the neutral lipid (NL) fatty acid composition of CHO cells cultured in the presence or absence of serum lipoproteins. In the presence of lipoproteins, ORP2/CHO cells display an increase in polyunsaturated PL species, and polyunsaturated fatty acids (PUFA) in the diminished NL pool are reduced. The increase of polyunsaturated PL may represent a compensatory response to alterations in cholesterol metabolism. Upon lipoprotein deprivation, the ORP2/CHO cells display a drop in polyunsaturated and an increase in mono and diunsaturated PL species. Our results suggest that this is due to defective recycling of PUFA from the diminished NL pool to PL. Furthermore, the PL PUFA, which are elevated in ORP2/CHO cells, are most likely subject to more rapid turnover than the NL-associated pool. The results provide evidence for a delicate integration of cholesterol, PL, and NL metabolism and a role of ORP2 as a regulator of the cellular lipidome.

  6. Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram; Kallehauge, Thomas Beuchert; Betenbaugh, Michael J; Nielsen, Alex Toftgaard; Kildegaard, Helene Faustrup

    2014-01-01

    Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:24827782

  7. Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool.

    PubMed

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Hansen, Henning Gram; Kallehauge, Thomas Beuchert; Betenbaugh, Michael J; Nielsen, Alex Toftgaard; Kildegaard, Helene Faustrup

    2014-08-01

    Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.

  8. Components of yeast (Sacchromyces cervisiae) extract as defined media additives that support the growth and productivity of CHO cells.

    PubMed

    Spearman, Maureen; Chan, Sarah; Jung, Vince; Kowbel, Vanessa; Mendoza, Meg; Miranda, Vivian; Butler, Michael

    2016-09-10

    Yeast and plant hydrolysates are used as media supplements to support the growth and productivity of CHO cultures for biopharmaceutical production. Through fractionation of a yeast lysate and metabolic analysis of a fraction that had bioactivity equivalent to commercial yeast extract (YE), bioactive components were identified that promoted growth and productivity of two recombinant CHO cell lines (CHO-Luc and CHO-hFcEG2) equivalent to or greater than YE-supplemented media. Autolysis of the yeast lysate was not necessary for full activity, suggesting that the active components are present in untreated yeast cells. A bioactive fraction (3KF) of the yeast lysate was isolated from the permeate using a 3kDa molecular weight cut-off (MWCO) filter. Supplementation of this 3KF fraction into the base media supported growth of CHO-Luc cells over eight passages equivalent to YE-supplemented media. The 3KF fraction was fractionated further by a cation exchange spin column using a stepwise pH elution. Metabolomic analysis of a bioactive fraction isolated at high pH identified several arginine and lysine-containing peptides as well as two polyamines, spermine and spermidine, with 3.5× and 4.5× higher levels compared to a fraction showing no bioactivity. The addition of a mixture of polyamines and their precursors (putrescine, spermine, spermidine, ornithine and citrulline) as well as increasing the concentration of some of the components of the original base medium resulted in a chemically-defined (CD) formulation that produced an equivalent viable cell density (VCD) and productivity of the CHO-Luc cells as the YE-supplemented medium. The VCD of the CHO-hFcEG2 culture in the CD medium was 1.9× greater and with equivalent productivity to the YE-supplemented media.

  9. Increased cellular internalization of amphiphiles in a multidrug-resistant CHO cell line.

    PubMed

    Boscoboinik, D; Epand, R M

    1989-10-30

    The uptake of labeled palmitoyl carnitine and palmitoyl lysophosphatidylcholine by CHO cells was studied by measuring the extractability of these amphiphiles by bovine serum albumin. A multidrug-resistant cell line, CHRC5, showed a more rapid uptake, compared with the parental line, of these amphiphiles into a pool that was no longer susceptible to extraction with bovine serum albumin. The more rapid uptake by the drug-resistant cell line was reversed back to the rates observed with the parental cell line in the presence of verapamil, quinacrine or cyclosporin A. These latter three drugs also reverse the multidrug-resistant phenotype. These results demonstrate a relationship between the rate of amphiphile uptake and multidrug resistance.

  10. Cytotoxic effects of zearalenone and its metabolites and antioxidant cell defense in CHO-K1 cells.

    PubMed

    Tatay, Elena; Font, Guillermina; Ruiz, Maria-Jose

    2016-10-01

    Zearalenone (ZEA) and its metabolites (α-zearalenol; α-ZOL, β-zearalenol; β-ZOL) are secondary metabolites of Fusarium fungi that produce cell injury. The present study explores mycotoxin-induced cell damage and cellular protection mechanisms in CHO-K1 cells. Cytotoxicity has been determined by reactive oxygen species (ROS) production and DNA damage. ROS production was determined using the fluorescein assay and DNA strand breakage by comet assay. Intracellular protection systems were glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). The results demonstrated that all mycotoxins increased the ROS levels up to 5.3-fold the control levels in CHO-K1 cells. Zearalenone metabolites, but not ZEA, increased DNA damage 43% (α-ZOL) and 28% (β-ZOL) compared to control cells. The GSH levels decreased from 18% to 36%. The GPx and SOD activities respectively increased from 26% to 62% and from 23% to 69% in CHO-K1 cells, whereas CAT activity decreased from 14% to 52%. In addition, intracellular ROS production was induced by ZEA and its metabolites. The endogenous antioxidant system components GSH, GPx and SOD were activated against ZEA and its metabolites. These antioxidant system components thus could contribute to decrease cell injury by ZEA and its metabolites.

  11. Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

    PubMed

    Zhang, An; Tsang, Valerie Liu; Markely, Lam R; Kurt, Lutfiye; Huang, Yao-Ming; Prajapati, Shashi; Kshirsagar, Rashmi

    2016-11-01

    In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

  12. The relationship between mTOR signalling pathway and recombinant antibody productivity in CHO cell lines

    PubMed Central

    2014-01-01

    Background High recombinant protein productivity in mammalian cell lines is often associated with phenotypic changes in protein content, energy metabolism, and cell growth, but the key determinants that regulate productivity are still not clearly understood. The mammalian target of rapamycin (mTOR) signalling pathway has emerged as a central regulator for many cellular processes including cell growth, apoptosis, metabolism, and protein synthesis. This role of this pathway changes in response to diverse environmental cues and allows the upstream proteins that respond directly to extracellular signals (such as nutrient availability, energy status, and physical stresses) to communicate with downstream effectors which, in turn, regulate various essential cellular processes. Results In this study, we have performed a transcriptomic analysis using a pathway-focused polymerase chain reaction (PCR) array to compare the expression of 84 target genes related to the mTOR signalling in two recombinant CHO cell lines with a 17.4-fold difference in specific monoclonal antibody productivity (q p ). Eight differentially expressed genes that exhibited more than a 1.5-fold change were identified. Pik3cd (encoding the Class 1A catalytic subunit of phosphatidylinositol 3-kinase [PI3K]) was the most differentially expressed gene having a 71.3-fold higher level of expression in the high producer cell line than in the low producer. The difference in the gene’s transcription levels was confirmed at the protein level by examining expression of p110δ. Conclusion Expression of p110δ correlated with specific productivity (q p ) across six different CHO cell lines, with a range of expression levels from 3 to 51 pg/cell/day, suggesting that p110δ may be a key factor in regulating productivity in recombinant cell lines. PMID:24533650

  13. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    PubMed

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line.

  14. Effects of copper on CHO cells: cellular requirements and product quality considerations.

    PubMed

    Yuk, Inn H; Russell, Stephen; Tang, Yun; Hsu, Wei-Ting; Mauger, Jacob B; Aulakh, Rigzen P S; Luo, Jun; Gawlitzek, Martin; Joly, John C

    2015-01-01

    Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes. © 2014 American Institute of Chemical Engineers.

  15. Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

    PubMed Central

    2012-01-01

    Background The possibility for isolating bovine mesenchymal multipotent cells (MSCs) from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D) environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM). The three-dimensional (3D) cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ) cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. Results Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when bovine-derived UC-WJ cells

  16. Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry.

    PubMed

    Ahn, Woo Suk; Antoniewicz, Maciek R

    2011-09-01

    Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-(13)C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC-MS) at 6, 12 and 24h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and (13)C-labeling dynamics of intracellular metabolites using non-stationary (13)C-metabolic flux analysis ((13)C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.

  17. Metabolic characterization of a CHO cell size increase phase in fed-batch cultures.

    PubMed

    Pan, Xiao; Dalm, Ciska; Wijffels, René H; Martens, Dirk E

    2017-09-26

    Normally, the growth profile of a CHO cell fed-batch process can be divided into two main phases based on changes in cell concentration, being an exponential growth phase and a stationary (non-growth) phase. In this study, an additional phase is observed during which the cell division comes to a halt but the cell growth continues in the form of an increase in cell size. The cell size increase (SI) phase occurs between the exponential proliferation phase (also called the number increase or NI phase) and the stationary phase. During the SI phase, the average volume and dry weight per cell increase threefold linearly with time. The average mAb specific productivity per cell increases linearly with the cell volume and therefore is on average two times higher in the SI phase than in the NI phase. The specific essential amino acids consumption rates per cell remain fairly constant between the NI and the SI phase, which agrees with the similar biomass production rate per cell between these two phases. Accumulation of fatty acids and formation of lipid droplets in the cells are observed during the SI phase, indicating that the fatty acids synthesis rate exceeds the demand for the synthesis of membrane lipids. A metabolic comparison between NI and SI phase shows that the cells with a larger size produce more mAb per unit of O2 and nutrient consumed, which can be used for further process optimization.

  18. Efficient Differentiation of Human Pluripotent Stem Cells into Mesenchymal Stem Cells by Modulating Intracellular Signaling Pathways in a Feeder/Serum-Free System

    PubMed Central

    Tran, Ngoc-Tung; Trinh, Quynh-Mai

    2012-01-01

    Mesenchymal stem cells (MSCs) derived from human pluripotent stem cells (hPSC-derived MSCs) will be one promising alternative cell source for MSC-based therapies. Here, an efficient protocol is demonstrated for generating hPSC-derived MSCs under a feeder-free culture system by regulating signaling pathways. Simultaneous treatments with Activin A, BIO (6-bromoindirubin-3′-oxime), and bone morphogenetic protein 4 (ABB) activated the transcription of mesoderm-lineage genes such as T, MIXL1, and WNT3 in hPSCs. The ABB-treated hPSCs could develop into CD105+ cells with a high efficiency of 20% in the MSC-induction medium. The properties of the hPSC-derived CD105+ cells were similar to those of adult MSCs in terms of surface antigens. Also, hPSC-derived MSCs had the potential to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro. The results demonstrated that functional MSCs could be generated efficiently from hPSCs by the combined modulation of signaling pathways. PMID:21793661

  19. Homologous desensitization of human histamine H₃ receptors expressed in CHO-K1 cells.

    PubMed

    Osorio-Espinoza, Angélica; Escamilla-Sánchez, Juan; Aquino-Jarquin, Guillermo; Arias-Montaño, José-Antonio

    2014-02-01

    Histamine H₃ receptors (H₃Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H₃Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H₃R (hH3R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [(35)S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [(3)H]-N-methyl-histamine ([(3)H]-NMHA) to intact cells and modified the sub-cellular distribution of H₃Rs, as detected by sucrose density gradients and [(3)H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl₂ and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH₃Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Serum-free bioprocessing of adult human and rodent skin-derived Schwann cells: implications for cell therapy in nervous system injury.

    PubMed

    Mirfeizi, Leila; Stratton, Jo Anne; Kumar, Ranjan; Shah, Prajay; Agabalyan, Natacha; Stykel, Morgan G; Midha, Rajiv; Biernaskie, Jeff; Kallos, Michael S

    2017-02-08

    Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

  1. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    PubMed

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc.

  2. Screening method for isolating DNA repair-deficient mutants of CHO cells

    SciTech Connect

    Thompson, L.H.; Rubin, J.S.; Cleaver, J.E.; Whitmore, G.F.; Brookman, K.

    1980-01-01

    A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (uv), ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two uv-sensitive clones studied in detail had a D/sub 37/ dose of 1.0 J/m/sup 2/ compared to 7.0 J/m/sup 2/ for the wild-type cells, and each was shown to have no detectable repair replication following exposure to uv doses of up to 26 J/m/sup 2/. Clones having hypersensitivity to alkylating agents, but not uv, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of /sup 60/Co ..gamma..-rays.

  3. Identification of CD9 extracellular domains important in regulation of CHO cell adhesion to fibronectin and fibronectin pericellular matrix assembly.

    PubMed

    Cook, George A; Longhurst, Celia M; Grgurevich, Svetozar; Cholera, Shila; Crossno, Joseph T; Jennings, Lisa K

    2002-12-15

    CD9, a 24-kDa member of the tetraspanin family, influences cellular growth and development, activation, adhesion, and motility. Our investigation focuses on the hypothesis that the CD9 second extracellular loop (EC2) is important in modulating cell adhesive events. Using a Chinese hamster ovary (CHO) cell expression system, we previously reported that CD9 expression inhibited cell adhesion to fibronectin and fibronectin matrix assembly. For the first time, a functional epitope on CD9 EC2 that regulates these processes is described. Binding of mAb7, an EC2-specific anti-CD9 monoclonal antibody, reversed the CD9 inhibitory activity on CHO cell adhesion and fibronectin matrix assembly. This reversal of cell phenotype also was observed in CHO cells expressing CD9 EC2 truncations. Furthermore, our data showed that the EC2 sequence (173)LETFTVKSCPDAIKEVFDNK(192) was largely responsible for the CD9-mediated CHO cell phenotype. Two peptides, (135)K-V(172) (peptide 5b) and (168)P-I(185) (peptide 6a), selectively blocked mAb7 binding to soluble CD9 and to CD9 on intact cells. These active peptides reversed the influence of CD9 expression on CHO cell adhesion to fibronectin. In addition, confocal microscopy revealed that CD9 colocalized with the integrin alpha(5)beta(1) and cytoskeletal F-actin in punctate clusters on the cell surface, particularly at the cell margins. Immunoprecipitation studies confirmed CD9 association with beta(1) integrin. The cellular distribution and colocalization of focal adhesion kinase and alpha-actinin with cytoskeletal actin was also influenced by CD9 expression. Thus, CD9 may exhibit its effect by modulating the composition of adhesive complexes important in facilitating cell adhesion and matrix assembly.

  4. Multigene expression in stable CHO cell pools generated with the piggyBac transposon system.

    PubMed

    Balasubramanian, Sowmya; Wurm, Florian M; Hacker, David L

    2016-09-01

    Heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB-derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB-derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB-derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308-1317, 2016. © 2016 American Institute of Chemical Engineers.

  5. Production of α2,6-sialylated IgG1 in CHO cells.

    PubMed

    Raymond, Céline; Robotham, Anna; Spearman, Maureen; Butler, Michael; Kelly, John; Durocher, Yves

    2015-01-01

    The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.

  6. Identification of a reactive glutathione conjugate as a metabolite of SR-2508 in CHO cells

    SciTech Connect

    Varghese, A.J.; Whitmore, G.F.

    1986-07-01

    Reaction between GSH and the hydroxylamine derivative of SR-2508 results in the formation of two stable conjugates identified as 2-amino-4-S-glutathionyl and 2-amino-5-S-glutathionyl imidazoles. These stable conjugates are apparently formed from a reactive derivative of the hydroxylamine that is sufficiently stable to be isolated after HPLC separation. The physical and chemical properties of this derivative are consistent with it being a GSH conjugate in which the glutathionyl residue is attached to the 2-amino nitrogen of the imidazole moiety through sulphur. With excess GSH, under physiological conditions, it forms a mixture of the two stable GSH conjugates. In CHO cells exposed to SR-2508 under hypoxic conditions, this unstable GSH conjugate has been detected and suggests the possibility of GSH functioning as a carrier of a toxic metabolite of 2-nitroimidazoles under certain conditions.

  7. Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system): enhancement by calcium phosphate

    SciTech Connect

    O'Neill, J.P.; Machanoff, R.; Sebastian, J.R.S.; Hsie, A.W.

    1982-01-01

    The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of a liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phosphate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range 0.3-3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagencity of DMN can be described as 110 mutants/10/sup 6/ cells per mM DMN per mg/ml S9 protein per hour.

  8. Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system): enhancement by calcium phosphate

    SciTech Connect

    O'Neill, J.P.; Machanoff, R.; San Sebastian, J.R.; Hsie, A.W.

    1982-01-01

    The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of a liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phospate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range 0.3-3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagenicity of DMN can be described as 110 mutants/10/sup 6/ cells per mM DMN per mg/ml S9 protein per hour.

  9. Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1.

    PubMed

    Jiang, Xiumei; Foldbjerg, Rasmus; Miclaus, Teodora; Wang, Liming; Singh, Rajinder; Hayashi, Yuya; Sutherland, Duncan; Chen, Chunying; Autrup, Herman; Beer, Christiane

    2013-09-12

    Investigation of the genotoxic potential of nanomaterials is essential to evaluate if they pose a cancer risk for exposed workers and consumers. The Chinese hamster ovary cell line CHO-K1 is recommended by the OECD for use in the micronucleus assay and is commonly used for genotoxicity testing. However, studies investigating if this cell line is suitable for the genotoxic evaluation of nanomaterials, including induction of DNA adduct and micronuclei formation, are rare and for silver nanoparticles (Ag NPs) missing. Therefore, we here systematically investigated DNA and chromosomal damage induced by BSA coated Ag NPs (15.9±7.6 nm) in CHO-K1 cells in relation to cellular uptake and intracellular localization, their effects on mitochondrial activity and production of reactive oxygen species (ROS), cell cycle, apoptosis and necrosis. Ag NPs are taken up by CHO-K1 cells and are presumably translocated into endosomes/lysosomes. Our cytotoxicity studies demonstrated a concentration-dependent decrease of mitochondrial activity and increase of intracellular reactive oxygen species (ROS) in CHO-K1 cells following exposure to Ag NPs and Ag⁺ (0-20 μg/ml) for 24h. Annexin V/propidium iodide assay showed that Ag NPs and Ag⁺ induced apoptosis and necrosis, which is in agreement with an increased fraction of cells in subG1 phase of the cell cycle. Genotoxicity studies showed that Ag NPs but also silver ions (Ag⁺) induced bulky-DNA adducts, 8-oxodG and micronuclei formation in a concentration-dependent manner, however, there were quantitative and qualitative differences between the particulate and ionic form of silver. Taken together, our multi-platform genotoxicity and cytotoxicity analysis demonstrates that CHO-K1 cells are suitable for the investigation of genotoxicity of nanoparticles like Ag NPs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production.

    PubMed

    Jimenez-Krassel, F; Ireland, J J

    2002-01-01

    The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells

  11. Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

    SciTech Connect

    Hsie, A.W.

    1980-01-01

    We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity.

  12. An in-silico study of the regulation of CHO cells glycolysis.

    PubMed

    Ghorbaniaghdam, Atefeh; Henry, Olivier; Jolicoeur, Mario

    2014-09-21

    In this work, a kinetic-metabolic model previously developed for CHO cells is used to study glycolysis regulation. The model is assessed for its biological relevance by analyzing its ability to simulate metabolic events induced following a hypoxic perturbation. Feedback and feedforward regulatory mechanisms known to occur to either inhibit or activate fluxes of glycolysis, are implemented in various combined scenarios and their effects on the metabolic response were analyzed. This study aims at characterizing the role of intermediates of glycolysis and of the cell energetic state, described as the AMP-to-ATP ratio, as inhibitors and activators of glycolysis pathway. In addition to the glycolysis pathway, we here describe the transient metabolic response of pathways that are connected to glycolysis, such as the pentose phosphate pathway, TCA cycle, cell bioenergetics system, glutamine and amino acids metabolisms. Taken individually, each regulatory mechanism leads to an oscillatory behavior in response to a hypoxic perturbation, while their combination clearly damps oscillations. However, only the addition of the cell energetic state to the regulatory mechanisms results in a non-oscillating response leading to metabolic flux rate rearrangement corresponding to the anaerobic metabolism expected to prevail under hypoxic conditions. We thus demonstrate in this work, from model simulations, that the robustness of a cell energetic metabolism can be described from a combination of feedback and feedforward inhibition and activation regulatory mechanisms of glycolysis fluxes, involving intermediates of glycolysis and the cell energetic state itself. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells.

    PubMed

    Kennard, M L; Food, M R; Jefferies, W A; Piret, J M

    1993-08-05

    A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.

  14. Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.

    PubMed

    Croset, Amelie; Delafosse, Laurence; Gaudry, Jean-Philippe; Arod, Christian; Glez, Loic; Losberger, Christophe; Begue, Damien; Krstanovic, Ana; Robert, Flavie; Vilbois, Francis; Chevalet, Laurent; Antonsson, Bruno

    2012-10-31

    Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. CHOPPI: a web tool for the analysis of immunogenicity risk from host cell proteins in CHO-based protein production.

    PubMed

    Bailey-Kellogg, Chris; Gutiérrez, Andres H; Moise, Leonard; Terry, Frances; Martin, William D; De Groot, Anne S

    2014-11-01

    Despite high quality standards and continual process improvements in manufacturing, host cell protein (HCP) process impurities remain a substantial risk for biological products. Even at low levels, residual HCPs can induce a detrimental immune response compromising the safety and efficacy of a biologic. Consequently, advanced-stage clinical trials have been cancelled due to the identification of antibodies against HCPs. To enable earlier and rapid assessment of the risks in Chinese Hamster Ovary (CHO)-based protein production of residual CHO protein impurities (CHOPs), we have developed a web tool called CHOPPI, for CHO Protein Predicted Immunogenicity. CHOPPI integrates information regarding the possible presence of CHOPs (expression and secretion) with characterizations of their immunogenicity (T cell epitope count and density, and relative conservation with human counterparts). CHOPPI can generate a report for a specified CHO protein (e.g., identified from proteomics or immunoassays) or characterize an entire specified subset of the CHO genome (e.g., filtered based on confidence in transcription and similarity to human proteins). The ability to analyze potential CHOPs at a genomic scale provides a baseline to evaluate relative risk. We show here that CHOPPI can identify clear differences in immunogenicity risk among previously validated CHOPs, as well as identify additional "risky" CHO proteins that may be expressed during production and induce a detrimental immune response upon delivery. We conclude that CHOPPI is a powerful tool that provides a valuable computational complement to existing experimental approaches for CHOP risk assessment and can focus experimental efforts in the most important directions. Biotechnol. Bioeng. 2014;111: 2170-2182. © 2014 Wiley Periodicals, Inc.

  16. miR-92a enhances recombinant protein productivity in CHO cells by increasing intracellular cholesterol levels.

    PubMed

    Loh, Wan Ping; Yang, Yuansheng; Lam, Kong Peng

    2017-04-01

    MicroRNAs (miRNAs) have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein productivity. Manipulation of miRNA levels in CHO cells have been shown to improve product yield by increasing proliferation and specific productivity (qP), resisting apoptosis and enhancing oxidative metabolism. The authors previously demonstrated that over-expressing miR-92a results in increases in qP and titer of CHO-IgG cells. However, the mechanisms by which miR-92a enhances qP in CHO cells are still uninvestigated. Here, the authors report the identification of insig1, a regulator of cholesterol biosynthesis, as a target of miR-92a using computational prediction. Both transient and stable over-expression of miR-92a decreased the expression levels of insig1. Insig1 was further validated as a target of miR-92a using 3' UTR reporter assay. Intracellular cholesterol concentration of two high-producing miR-92a clones were significantly increased by ≈30% compared to the blank-transfected pool. Relative Golgi surface area was also found to be 18-26% higher in these clones. Our findings suggest that miR-92a may affect cholesterol metabolism by repressing insig1, resulting in raised intracellular cholesterol levels and Golgi volume and hence enhanced protein secretion. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Effects of CHO-expressed recombinant lactoferrins on mouse dendritic cell presentation and function.

    PubMed

    Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

    2015-07-01

    Lactoferrin (LF), a natural iron-binding protein, has previously demonstrated effectiveness in enhancing the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine. This report investigates immune modulatory effects of Chinese hamster ovary (CHO) cell-expressed recombinant mouse and human LFs on mouse bone marrow-derived dendritic cells (BMDCs), comparing homologous and heterologous functions. BCG-infected BMDCs were cultured with LF, and examined for class II presentation molecule expression. Culturing of BCG-infected BMDCs with either LF decreased the class II molecule-expressing population. Mouse LF significantly increased the production of IL-12p40, IL-1β and IL-10, while human LF-treated BMDCs increased only IL-1β and IL-10. Overlaying naïve CD4 T-cells onto BCG-infected BMDCs cultured with mouse LF increased IFN-γ, whereas the human LF-exposed group increased IFN-γ and IL-17 from CD4 T cells. Overlay of naïve CD8 T cells onto BCG-infected BMDCs treated with mouse LF increased the production of IFN-γ and IL-17, while similar experiments using human LF only increased IL-17. This report is the first to examine mouse and human recombinant LFs in parallel experiments to assess murine DC function. These results detail the efficacy of the human LF counterpart used in a heterologous system to understand LF-mediated events that confer BCG efficacy against Mycobacterium tuberculosis challenge.

  18. Response of CHO cell proliferation and histone phosphorylation to sodium arsenite

    SciTech Connect

    Gurley, L.R.; Walters, R.A.; Jett, J.H.; Tobey, R.A.

    1980-01-01

    Because of the causal association between arsenic and cancer in humans, studies were initiated to determine whether low levels of arsenic affect cell proliferation and chromatin constituents. Cultured CHO cells were treated with As(III) or As(V) in the form of sodium arsenite (NaAsO/sub 2/) or sodium arsenate (Na/sub 2/HAsO/sub 4/), respectively. Arsenite was 10 times more toxic than arsenate. At lower concentrations, 10 ..mu..M NaAsO/sub 2/ induced culture growth kinetics that were radiomimetic. During this division delay, histone Hl phosphorylation was reduced to 40% of the control level. It recovered to the control level just before recovery of cell division. Histone H2A phosphorylation was stimulated in the middle of the delay period, followed by stimulation of H3 phosphorylation just before recovery of cell division. ATP pool levels were unaffected at these low arsenite concentrations. The DNA synthesis rate was also reduced during division delay, but it did not recover immediately before resumption of cell division. Flow microfluorometric analysis showed that cell cycle progression was inhibited in G/sub 1/, S, and G/sub 2/ during the division delay period. On recovery of cell division, cells in S and G/sub 2/ regained the capacity to resume cycle traverse earlier than did cells in G/sub 1/. Cell survival studies indicated that 89% of the cells survived 24-h treatment with 10 ..mu..M NaAsO/sub 2/.

  19. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    PubMed

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  20. Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

    PubMed

    Ghasemi-Dehkordi, Payam; Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Mirzaeian, Amin; Saffari-Chaleshtori, Javad; Heybati, Fatemeh; Mardani, Gashtasb; Karimi-Taghanaki, Alireza; Doosti, Abbas; Jami, Mohammad-Saeid; Abolhasani, Marziyeh; Hashemzadeh-Chaleshtori, Morteza

    2015-09-01

    Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens

  1. Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

    PubMed

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Serra, Elisa; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2016-12-20

    Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 10(6) cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 10(6) cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  2. Analyzing Clonal Variation of Monoclonal Antibody-Producing CHO Cell Lines Using an In Silico Metabolomic Platform

    PubMed Central

    Ghorbaniaghdam, Atefeh; Chen, Jingkui; Henry, Olivier; Jolicoeur, Mario

    2014-01-01

    Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones. PMID

  3. A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs

    PubMed Central

    Salvagiotto, Giorgia; Burton, Sarah; Daigh, Christine A.; Rajesh, Deepika; Slukvin, Igor I.; Seay, Nicholas J.

    2011-01-01

    Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells. PMID:21445267

  4. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.

    PubMed Central

    Israel, D I; Kaufman, R J

    1989-01-01

    A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion. Images PMID:2546123

  5. Role of the Nfa1 protein in pathogenic Naegleria fowleri cocultured with CHO target cells.

    PubMed

    Kang, Su-Yeon; Song, Kyoung-Ju; Jeong, Seok-Ryoul; Kim, Jong-Hyun; Park, Sun; Kim, Kyongmin; Kwon, Myung-Hee; Shin, Ho-Joon

    2005-07-01

    Naegleria fowleri, a free-living amoeba, exists as a virulent pathogen which causes fatal primary amoebic meningoencephalitis in experimental animals and humans. Using infected and immune mouse sera, we previously cloned an nfa1 gene from a cDNA library of N. fowleri by immunoscreening. The nfa1 gene (360 bp) produced a recombinant 13.1-kDa protein, and the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. In this study, the role of the Nfa1 protein as a cell contact mechanism of N. fowleri cocultured with target cells was observed by an immunofluorescence assay with an anti-Nfa1 polyclonal antibody. Using confocal microscopic findings, the Nfa1 protein was located on the pseudopodia of N. fowleri trophozoites. The Nfa1 protein in N. fowleri trophozoites cocultured with CHO target cells was also located on pseudopodia, as well as in a food cup formed as a phagocytic structure in close contact with target cells. The amount of nfa1 mRNA of N. fowleri was strongly increased 6 h after coculture.

  6. A kinetic-metabolic model based on cell energetic state: study of CHO cell behavior under Na-butyrate stimulation.

    PubMed

    Ghorbaniaghdam, Atefeh; Henry, Olivier; Jolicoeur, Mario

    2013-04-01

    A kinetic-metabolic model approach describing and simulating Chinese hamster ovary (CHO) cell behavior is presented. The model includes glycolysis, pentose phosphate pathway, TCA cycle, respiratory chain, redox state and energetic metabolism. Growth kinetic is defined as a function of the major precursors for the synthesis of cell building blocks. Michaelis-Menten type kinetic is used for metabolic intermediates as well as for regulatory functions from energy shuttles (ATP/ADP) and cofactors (NAD/H and NADP/H). Model structure and parameters were first calibrated using results from bioreactor cultures of CHO cells expressing recombinant t-PA. It is shown that the model can simulate experimental data for all available experimental data, such as extracellular glucose, glutamine, lactate and ammonium concentration time profiles, as well as cell energetic state. A sensitivity analysis allowed identifying the most sensitive parameters. The model was then shown to be readily adaptable for studying the effect of sodium butyrate on CHO cells metabolism, where it was applied to the cases with sodium butyrate addition either at mid-exponential growth phase (48 h) or at the early plateau phase (74 h). In both cases, a global optimization routine was used for the simultaneous estimation of the most sensitive parameters, while the insensitive parameters were considered as constants. Finally, confidence intervals for the estimated parameters were calculated. Results presented here further substantiate our previous findings that butyrate treatment at mid-exponential phase may cause a shift in cellular metabolism toward a sustained and increased efficiency of glucose utilization channeled through the TCA cycle.

  7. Verhulst and stochastic models for comparing mechanisms of MAb productivity in six CHO cell lines.

    PubMed

    Shirsat, Nishikant; Avesh, Mohd; English, Niall J; Glennon, Brian; Al-Rubeai, Mohamed

    2016-08-01

    The present study validates previously published methodologies-stochastic and Verhulst-for modelling the growth and MAb productivity of six CHO cell lines grown in batch cultures. Cytometric and biochemical data were used to model growth and productivity. The stochastic explanatory models were developed to improve our understanding of the underlying mechanisms of growth and productivity, whereas the Verhulst mechanistic models were developed for their predictability. The parameters of the two sets of models were compared for their biological significance. The stochastic models, based on the cytometric data, indicated that the productivity mechanism is cell specific. However, as shown before, the modelling results indicated that G2 + ER indicate high productivity, while G1 + ER indicate low productivity, where G1 and G2 are the cell cycle phases and ER is Endoplasmic Reticulum. In all cell lines, growth proved to be inversely proportional to the cumulative G1 time (CG1T) for the G1 phase, whereas productivity was directly proportional to ER. Verhulst's rule, "the lower the intrinsic growth factor (r), the higher the growth (K)," did not hold for growth across all cell lines but held good for the cell lines with the same growth mechanism-i.e., r is cell specific. However, the Verhulst productivity rule, that productivity is inversely proportional to the intrinsic productivity factor (r x ), held well across all cell lines in spite of differences in their mechanisms for productivity-that is, r x is not cell specific. The productivity profile, as described by Verhulst's logistic model, is very similar to the Michaelis-Menten enzyme kinetic equation, suggesting that productivity is more likely enzymatic in nature. Comparison of the stochastic and Verhulst models indicated that CG1T in the cytometric data has the same significance as r, the intrinsic growth factor in the Verhulst models. The stochastic explanatory and the Verhulst logistic models can explain the

  8. Unique gene expression signature by human embryonic stem cells cultured under serum-free conditions correlates with their enhanced and prolonged growth in an undifferentiated stage.

    PubMed

    Skottman, Heli; Strömberg, Anne-Marie; Matilainen, Eija; Inzunza, Jose; Hovatta, Outi; Lahesmaa, Riitta

    2006-01-01

    Understanding the interaction between human embryonic stem cells (hESCs) and their microenvironment is crucial for the propagation and the differentiation of hESCs for therapeutic applications. hESCs maintain their characteristics both in serum-containing and serum-replacement (SR) media. In this study, the effects of the serum-containing and SR culture media on the gene expression profiles of hESCs were examined. Although the expression of many known embryonic stem cell markers was similar in cells cultured in either media, surprisingly, 1,417 genes were found to be differentially expressed when hESCs cultured in serum-containing medium were compared with those cultured in SR medium. Several genes upregulated in cells cultured in SR medium suggested increased metabolism and proliferation rates in this medium, providing a possible explanation for the increased growth rate of nondifferentiated cells observed in SR culture conditions compared with that in serum medium. Several genes characteristic for cells with differentiated phenotype were expressed in cells cultured in serum-containing medium. Our data clearly indicate that the manipulation of hESC culture conditions causes phenotypic changes of the cells that were reflected also at the level of gene expression. Such changes may have fundamental importance for hESCs, and gene expression changes should be monitored as a part of cell culture optimization aiming at a clinical use of hESCs for cell transplantation.

  9. Mutagenicity of silver nanoparticles in CHO cells dependent on particle surface functionalization and metabolic activation

    NASA Astrophysics Data System (ADS)

    Guigas, Claudia; Walz, Elke; Gräf, Volker; Heller, Knut J.; Greiner, Ralf

    2017-06-01

    The potential of engineered nanomaterials to induce genotoxic effects is an important aspect of hazard identification. In this study, cytotoxicity and mutagenicity as a function of metabolic activation of three silver nanoparticle (AgNP) preparations differing in surface coating were determined in Chinese hamster ovary (CHO) subclone K1 cells. Three silver nanoparticle preparations ( x 90,0 <30 nm) stabilized with polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate (AgPure™), citrate (Citrate-Ag), and polyvinylpyrrolidone (PVP-Ag) were used for the experiments. The cytotoxic effect of AgNPs was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) test using different concentrations of nanoparticles, while the mutagenicity was evaluated using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. The cytotoxicity of all three AgNPs was lower in a cell culture medium containing 10% fetal calf serum (FCS) than in medium without FCS. The HPRT test without metabolic activation system S9 revealed that compared to the other AgNP formulations, citrate-coated Ag showed a lower genotoxic effect. However, addition of S9 increased the mutation frequency of all AgNPs and especially influenced the genotoxicity of Citrate-Ag. The results showed that exogenous metabolic activation of nanosilver is crucial even if interactions of the metabolic activation system, nanosilver, and cells are not really understood up to now.

  10. Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia.

    PubMed

    Cole, A; Armour, E P

    1988-09-01

    A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.

  11. Microparticle-Mediated Transfer of the Viral Receptors CAR and CD46, and the CFTR Channel in a CHO Cell Model Confers New Functions to Target Cells

    PubMed Central

    Gonzalez, Gaëlle; Vituret, Cyrielle; Di Pietro, Attilio; Chanson, Marc; Boulanger, Pierre; Hong, Saw-See

    2012-01-01

    Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins. PMID:23284987

  12. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    PubMed

    Gonzalez, Gaëlle; Vituret, Cyrielle; Di Pietro, Attilio; Chanson, Marc; Boulanger, Pierre; Hong, Saw-See

    2012-01-01

    Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  13. Cell alterations but no DNA strand breaks induced in vitro by cylindrospermopsin in CHO K1 cells.

    PubMed

    Fessard, Valérie; Bernard, Cécile

    2003-10-01

    Cylindrospermopsin has been involved in some cyanobacterial blooms associated with animal and human intoxications in different countries. Liver is the main target organ even though thymus and kidney are also affected. Its toxic effect has been shown to be induced by protein synthesis inhibition. However, further research about its toxicological potential is required, as revealed by the U.S. Unregulated Contaminant Monitoring Regulation (US UCMR) meeting in 2001. Induction of DNA damage by cylindrospermopsin has been reported by some authors either by a direct effect on DNA or by an indirect effect on associated macromolecules. This study focused on evaluating its in vitro genotoxic potential using the comet assay coupled to various cell alteration measurements. No DNA damage was detected by the alkaline comet assay on Chinese hamster ovary (CHO) K1 cells after 24 h of treatment with cylindrospermopsin concentrations of 0.5 and 1 microg mL(-1). However, inhibition of cell growth was noticed as well as cell blebbing and rounding. These morphological effects were linked to cytoskeletal reorganization (mainly microfilaments) but not to apoptosis. This study concluded that cylindrospermopsin does not obviously react directly with DNA in CHO K1 cells. But the hypothesis of its metabolization into a genotoxic product must be explored further.

  14. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer?

    PubMed

    Chusainow, Janet; Yang, Yuan Sheng; Yeo, Jessna H M; Toh, Poh Choo; Asvadi, Parisa; Wong, Niki S C; Yap, Miranda G S

    2009-03-01

    Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize

  15. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    SciTech Connect

    Schwartz, J.L.; Sun, J.; Porter, R.C.

    1995-11-01

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, {sup 60}Co x-ray-, and {sup 212}Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of {gamma} rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from {sup 212}Bi, a {sup 220}Rn daughter, resulted in a spectrum similar to the {gamma}-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The {alpha}-radiation, however, tended to produce larger intragenic deletions that {gamma} radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to {alpha} radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect {alpha}-induced mutation spectra.

  16. Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

    PubMed

    Betts, Zeynep; Dickson, Alan J

    2016-03-01

    The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

  17. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody.

    PubMed

    Carrillo-Cocom, L M; Genel-Rey, T; Araíz-Hernández, D; López-Pacheco, F; López-Meza, J; Rocha-Pizaña, M R; Ramírez-Medrano, A; Alvarez, M M

    2015-10-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.

  18. High-frequency deletion event at aprt locus of CHO cells: detection and characterization of endpoints.

    PubMed

    Dewyse, P; Bradley, W E

    1989-01-01

    Two mechanisms are implicated in generating recessive drug resistance mutants at the adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary (CHO) cells, one of which is a spontaneous high-frequency deletion of the entire gene. We have isolated and mapped a 19-kb fragment carrying aprt and its flanking sequences. A Southern blot study of 198 independent deletion mutants revealed that two different mutants have one of their breakpoints within the 19-kb region analyzed. One of these has an upstream breakpoint which could be narrowed down to a 4-kb fragment containing repetitive sequences. The other mutant has a breakpoint within a 410-bp sequence located 8.5 kb downstream of the aprt gene and which carries several elements similar to those signaling V-(D)-J joining in immunoglobulin and T-cell receptor gene rearrangements. In each case the other breakpoint lay outside of the analyzed region. These results support the previous indications that the deletions created by this spontaneous event are large.

  19. Overexpression of heat shock proteins (HSPs) in CHO cells for extended culture viability and improved recombinant protein production.

    PubMed

    Lee, Yih Yean; Wong, Kathy T K; Tan, Janice; Toh, Poh Choo; Mao, Yanying; Brusic, Vesna; Yap, Miranda G S

    2009-08-10

    It has been widely reported that CHO cells undergo apoptosis in culture, despite supplementation of nutrients through fed-batch strategies. Improvement of cell viability in culture can effectively improve recombinant protein yield through extension of the culture's production lifespan, especially at high cell densities. Heat shock proteins (HSPs) have been reported to demonstrate anti-apoptotic effects against a wide range of physical and chemical stimuli through their ability to bind and act as antagonists to critical apoptotic molecules. CHO-IFN-gamma cells, expressing recombinant human interferon-gamma (IFN-gamma), were engineered to overexpress two HSPs (HSP27 and HSP70) either individually or in combination. In fed-batch bioreactor cultures, the engineered cell lines exhibited a more gradual viability loss and extension of culture times of 36-72h, with corresponding delays in escalation of caspases 2, 3, 8 and 9 activities, compared to the control cultures utilizing cells transfected with the vector backbone. The extension in culture times translated to a 2.5-fold improvement in IFN-gamma production over controls in fed-batch cultures. These results suggest that overexpression of HSPs represents a promising generic strategy for the development of robust CHO cell lines resistant to apoptotic insults and possessing improved culture characteristics to enhance recombinant glycoprotein yields.

  20. Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures.

    PubMed

    Schallmoser, Katharina; Strunk, Dirk

    2009-10-30

    Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo(1,2) and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin(3-5) or lysed by freeze/thaw cycles(6-14) before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.(1,15-17) Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors(18,19) and a current lack of standardized protocols for platelet preparation.(15,16) This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

  1. The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures.

    PubMed

    Hyoung Park, Jin; Sin Lim, Myung; Rang Woo, Ju; Won Kim, Jong; Min Lee, Gyun

    2016-09-01

    To investigate the effect of dextran sulfate (DS), a widely used anti-aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM-0.025 and CS13-1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13-1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13-1.00 cells, and showed the highest maximum mAb concentration. For SM-0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N-glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13-1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3-1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113-1122, 2016. © 2016 American Institute of Chemical Engineers.

  2. Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

    PubMed Central

    2010-01-01

    Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco’s modified Eagle’s medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (106 cells/mL) of P1 ASCs were frozen overnight in a −80°C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37°C water bath (1–2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37°C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, ∼84% ± 5% and ∼84% ± 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM. PMID:19788372

  3. Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.

    PubMed

    Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; Götze, Marco; Felka, Tino; Aicher, Wilhelm K; Kluba, Torsten

    2012-11-02

    Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

  4. Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

    PubMed Central

    Mittag, Falk; Falkenberg, Eva-Maria; Janczyk, Alexandra; Götze, Marco; Felka, Tino; Aicher, Wilhelm K.; Kluba, Torsten

    2012-01-01

    Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1–5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications. PMID:23589764

  5. Lucifer Yellow uptake by CHO cells exposed to magnetic and electric pulses

    PubMed Central

    Towhidi, Leila; Firoozabadi, Seyed Mohammad P; Mozdarani, Hossein; Miklavcic, Damijan

    2012-01-01

    Background The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Due to numerous applications in medicine, biology and biotechnology, the introduction of impermeant molecules into biological cells has drawn considerable attention in the past years. One of the most famous methods in this field is electroporation, in which electric pulses with high intensity and short duration are applied to the cells. The aim of our study was to investigate the effect of time-varying magnetic field with different parameters on transmembrane molecular transport. Materials and methods. ‘Moreover, a comparison was made between the uptake results due to magnetic pulse exposure and electroporation mediated uptake.’ at the end of Background part. The Chinese hamster ovary (CHO) cells were exposed to magnetic pulses of 2.2 T peak strength and 250 μs duration delivered by Magstim stimulator and double 70 mm coil. Three different frequencies of 0.25, 1 and 10 Hz pulses with 112, 56 and 28 number of pulses were applied (altogether nine experimental groups) and Lucifer Yellow uptake was measured in each group. Moreover, maximum uptake of Lucifer Yellow obtained by magnetic pulses was compared to the measured uptake due to electroporation with typical parameters of 8 pulses of 100 μs, repetition frequency of 1 Hz and electric field intensities of 200 to 600 V/cm. Results and conclusions. Our results show that time-varying magnetic field exposure increases transmembrane molecular transport and this uptake is greater for lower frequencies and larger number of pulses. Besides, the comparison shows that electroporation is more effective than pulsed magnetic field, but the observed uptake enhancement due to magnetic exposure is still considerable. PMID:23077448

  6. Lucifer Yellow uptake by CHO cells exposed to magnetic and electric pulses.

    PubMed

    Towhidi, Leila; Firoozabadi, Seyed Mohammad P; Mozdarani, Hossein; Miklavcic, Damijan

    2012-06-01

    The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Due to numerous applications in medicine, biology and biotechnology, the introduction of impermeant molecules into biological cells has drawn considerable attention in the past years. One of the most famous methods in this field is electroporation, in which electric pulses with high intensity and short duration are applied to the cells. The aim of our study was to investigate the effect of time-varying magnetic field with different parameters on transmembrane molecular transport. 'Moreover, a comparison was made between the uptake results due to magnetic pulse exposure and electroporation mediated uptake.' at the end of Background part. The Chinese hamster ovary (CHO) cells were exposed to magnetic pulses of 2.2 T peak strength and 250 μs duration delivered by Magstim stimulator and double 70 mm coil. Three different frequencies of 0.25, 1 and 10 Hz pulses with 112, 56 and 28 number of pulses were applied (altogether nine experimental groups) and Lucifer Yellow uptake was measured in each group. Moreover, maximum uptake of Lucifer Yellow obtained by magnetic pulses was compared to the measured uptake due to electroporation with typical parameters of 8 pulses of 100 μs, repetition frequency of 1 Hz and electric field intensities of 200 to 600 V/cm. Our results show that time-varying magnetic field exposure increases transmembrane molecular transport and this uptake is greater for lower frequencies and larger number of pulses. Besides, the comparison shows that electroporation is more effective than pulsed magnetic field, but the observed uptake enhancement due to magnetic exposure is still considerable.

  7. Phyllanthus orbicularis aqueous extract: cytotoxic, genotoxic, and antimutagenic effects in the CHO cell line.

    PubMed

    Sànchez-Lamar, A; Fiore, M; Cundari, E; Ricordy, R; Cozzi, R; De Salvia, R

    1999-12-15

    The present work evaluates the cytotoxic, genotoxic, and antimutagenic effects of Phyllanthus orbicularis (plant of genus Phyllantus) aqueous extract in Chinese hamster ovary (CHO) cells. P. orbicularis aqueous extracts are used in Cuban traditional medicine for their antiviral activity against Hepatitis B virus and A and B flu virus. The cytotoxicity of the extract was tested by means of colony-forming ability and growth-inhibition assays as well as by measuring the mitotic index. Apoptosis induction and cell-cycle kinetics were analyzed by cytofluorimetric methods. Chromosome aberration assays were performed to study the genotoxic and antimutagenic activity of the extract. Results show that doses of up to 100 microg/ml of the extract did not induce any cytotoxic effects. Cell survival and mitotic index decreased significantly at doses higher than 100 microg/ml as a function of dose as well as of treatment time. Moreover, continuous treatments of up to 18 h induced the appearance of a significant number of apoptotic cells. Following a 3-h exposure to a dose of 750 microg/ml, cells accumulated significantly in G(2)-M phase and remained blocked in G(1-) and G(2)-M phases after several posttreatments in fresh growth medium. The aqueous extract alone did not induce chromosome aberrations but, in combined treatment with H(2)O(2), significantly reduced H(2)O(2)-induced chromosome aberrations. Flow cytometric analysis of DCFH intracellular oxidation showed that the extract decreased the oxidizing power of H(2)O(2.) This ability could possibly explain the extract's antigenotoxic activity. Absence of cytotoxicity at the lower tested doses and the antimutagenic properties of the extract stimulate the interest in studying possible new pharmaceutical uses of P. orbicularis.

  8. CHO cells knocked out for TSC2 display an improved productivity of antibodies under fed batch conditions.

    PubMed

    McVey, Duncan; Aronov, Michael; Rizzi, Giovanni; Cowan, Alexis; Scott, Charo; Megill, John; Russell, Reb; Tirosh, Boaz

    2016-09-01

    The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    PubMed

    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.

  10. Homodimerization and internalization of galanin type 1 receptor in living CHO cells.

    PubMed

    Wirz, Sebastian A; Davis, Christopher N; Lu, Xiaoying; Zal, Tomasz; Bartfai, Tamas

    2005-12-01

    Galanin is a 29- to 30-aa-long neuropeptide affecting feeding, cognitive, and sexual behavior. It exerts its effects through galanin receptors 1, 2 and 3, which are all seven transmembrane domain G-protein coupled receptors (GPCRs). The GPCRs have been shown to function as monomers, homodimers, heterodimers and oligomers. In this study, we examined the extent of galanin receptor 1 (GalR1) dimerization and internalization in living CHO cells using fluorescence resonance energy transfer (FRET) and time lapse confocal imaging. Ratio imaging analysis and emission spectral analysis revealed substantial homodimerization of GalR1. In addition, internalization of GalR1 after 1h of agonist stimulation with the GalR1 agonist galanin (1-29) was observed with time lapse fluorescence imaging, whereas stimulation with the GalR2 specific agonist galanin (2-11) did not lead to internalization. Treatment of GalR1 transfected cells with the non-selective adenylyl cyclase activator forskolin influenced the rate of internalization when administered together with galanin (1-29). These results indicate that GalR1 can act as a dimer on the cell surface and that receptor desensitization and internalization was observed after stimulation with the agonist galanin (1-29). Western blots further confirm the FRET data that GalR1-XFP dimerizes and can be detected in the cell as a monomer or dimer using antibodies to XFP. Internalization and dimerization of GalR1 is shown, contributing to the regulation of galanergic signaling.

  11. Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A

    PubMed Central

    1994-01-01

    22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle- specific coats. PMID:8027187

  12. A synbiotic containing Lactobacillus gasseri [corrected] CHO-220 and inulin improves irregularity of red blood cells.

    PubMed

    Ooi, L-G; Bhat, R; Rosma, A; Yuen, K-H; Liong, M-T

    2010-10-01

    This randomized, double-blind, placebo-controlled, and parallel-design study was conducted to investigate the effect of a synbiotic product containing Lactobacillus gasseri [corrected] CHO-220 and inulin on the irregularity in shape of red blood cells (RBC) in hypercholesterolemic subjects. The subjects (n=32) were randomly allocated to 2 groups, a treatment group (synbiotic product) and a control group (placebo), and received 4 capsules of either synbiotic or placebo daily for 12 wk. Morphological representation via scanning electron microscopy showed that the occurrence of spur RBC was improved upon supplementation of the synbiotic. In addition, the supplementation of synbiotic reduced the cholesterol:phospholipids ratio of the RBC membrane by 47.02% over 12 wk, whereas the control showed insignificant changes. Our present study also showed that supplementation of the synbiotic reduced the concentration of saturated fatty acids (SFA), increased unsaturated fatty acids (UFA), and increased the ratio of UFA:SFA over 12 wk, whereas the control showed inconspicuous changes. The alteration of RBC membrane was assessed using fluorescence anisotropy (FAn) and fluorescence probes with different affinities for varying sections of the membrane phospholipid bilayer. A noticeable decrease in FAn of three fluorescent probes was observed in the synbiotic group compared with the control over 12 wk, indicative of increased membrane fluidity and reduced cholesterol enrichment in the RBC membrane. Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Sewage sludge hazardous assessment: chemical evaluation and cytological effects in CHO-k1 cells.

    PubMed

    Bonomo, M M; Morozesk, M; Duarte, I D; Rocha, L D; Fernandes, M N; Matsumoto, S T

    2016-06-01

    Application of sewage sludge in agricultural lands is a growing practice in several countries due to its numerous benefits to soil and crops, where chemical and pathogen levels are determined by corresponding legislation. However, the presence of contaminants in residues must always be controlled before application due to their dangerous effects over the ecosystem and potential risks to human health. The main objective of this study was to integrate biological and chemical analysis in order to help elucidating the residue potential toxic, cytotoxic, and mutagenic effects. We evaluate samples of sewage sludge before and after the sanitizing treatment with lime in cytokinesis-block assay using CHO-k1 culture cells. The sanitizing treatment promoted a decrease in pathogen levels, which is the main purpose of this process. Even with chemical levels below the established by environmental agencies, results showed sewage sludge ability to enhance genotoxic and mutagenic effects, proving that residue should be handled with caution in order to minimize its environmental and human risk.

  14. "Salt tolerant" anion exchange chromatography for direct capture of an acidic protein from CHO cell culture.

    PubMed

    Champagne, Jérôme; Balluet, Guillaume; Gantier, René; Toueille, Magali

    2013-06-01

    The present study describes the use of the new HyperCel STAR AX "salt tolerant" anion exchange sorbent for the capture from Chinese Hamster Ovary (CHO) cell culture supernatant (CCS) of an acidic model protein (α-amylase). HyperCel STAR AX sorbent and other conventional anion exchangers were evaluated to purify biologically-active enzyme. Salt tolerance of the sorbent allowed reaching 5-fold higher dynamic binding capacity than conventional anion exchange during capture of the enzyme from neat (undiluted) CCS. After optimization of operating conditions, HyperCel STAR AX turned out to be the only sorbent allowing efficient protein capture directly from both neat and diluted CCS with consistent and satisfying purity, yield and productivity. Therefore implementation of the salt tolerant sorbent in industrial purification processes should allow avoiding time and cost consuming steps such as dilution or UF/DF that exclusively aim at establishing suitable conditions for ion exchange step without bringing any added value to the purification process performance. Altogether this study highlights the flexibility and cost-reduction potential brought in process design by the HyperCel STAR AX salt tolerant sorbent.

  15. High-Throughput Lipidomic and Transcriptomic Analysis To Compare SP2/0, CHO, and HEK-293 Mammalian Cell Lines.

    PubMed

    Zhang, Yue; Baycin-Hizal, Deniz; Kumar, Amit; Priola, Joseph; Bahri, Michelle; Heffner, Kelley M; Wang, Miao; Han, Xianlin; Bowen, Michael A; Betenbaugh, Michael J

    2017-02-07

    A combined lipidomics and transcriptomics analysis was performed on mouse myeloma SP2/0, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells in order to compare widely used mammalian expression systems. Initial thin layer chromatography (TLC) analysis indicated that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major lipid components in all cell lines with lower amounts of sphingomyelin (SM) in SP2/0 compared to CHO and HEK, which was subsequently confirmed and expanded upon following mass spectrometry (MS) analysis. HEK contained 4-10-fold higher amounts of lyso phosphatidylethanolamine (LPE) and 2-4-fold higher amounts of lyso phosphatidylcholine (LPC) compared to SP2/0 and CHO cell lines. C18:1 followed by C16:1 were the main contributors to the difference in both LPE and LPC levels. Alternatively, the SP2/0 cell line exhibited 30-65-fold lower amounts of SM principally in the amount of 16:0. By mapping the transcriptomics data to KEGG pathways, we found expression levels of secretory phospholipase A2 (sPLA2), lysophospholipid acyltransferase (LPEAT), lysophosphatidylcholine acyltransferase (LPCAT), and lysophospholipase (LYPLA) can contribute to the differences in LPE and LPC. Sphingomyelin synthases (SMS) and sphingomyelin phosphodiesterase (SMase) enzymes may play roles in SM differences across the three cell lines. The results of this study provide insights that will aid the understanding of the physiological and secretory differences across recombinant protein production systems.

  16. C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.

    PubMed

    Otsuka, Ryo; Harada, Nagakatsu; Aoki, Shouhei; Shirai, Kanna; Nishitsuji, Kazuchika; Nozaki, Ayane; Hatakeyama, Adzumi; Shono, Masayuki; Mizusawa, Noriko; Yoshimoto, Katsuhiko; Nakaya, Yutaka; Kitahata, Hiroshi; Sakaue, Hiroshi

    2016-01-01

    GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.

  17. Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines.

    PubMed

    Balasubramanian, Sowmya; Rajendra, Yashas; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-06-01

    Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc.

  18. A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

    PubMed

    Rajendra, Yashas; Hougland, Maria D; Alam, Riazul; Morehead, Teresa A; Barnard, Gavin C

    2015-05-01

    Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers

  19. Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells.

    PubMed

    Coryell, Virginia H; Stearns, Diane M

    2006-11-07

    Chromium picolinate (CrPic) is a popular dietary supplement, marketed to the public for weight loss, bodybuilding, and control of blood sugar. Recommendations for long-term use at high dosages have led to questions regarding its safety. Previous studies have reported that CrPic can cause chromosomal aberrations and mutations. The purpose of the current work was to compare the mutagenicity of CrPic as a suspension in acetone versus a solution in DMSO, and to characterize the hprt mutations induced by CrPic in CHO AA8 cells. Treatments of 2% acetone or 2% DMSO alone produced no significant increase in 6-thioguanine (6-TG)-resistant mutants after 48 h exposures. Mutants resistant to 6-TG were generated by exposing cells for 48 h to 80 microg/cm(2) CrPic in acetone or to 1.0mM CrPic in DMSO. CrPic in acetone produced an average induced mutation frequency (MF) of 56 per 10(6) surviving cells relative to acetone solvent. CrPic in acetone was 3.5-fold more mutagenic than CrPic in DMSO, which produced an MF of 16.2. Characterization of 61 total mutations in 48 mutants generated from exposure to CrPic in acetone showed that base substitutions comprised 33% of the mutations, with transversions being predominant; deletions made up 62% of the mutations, with one-exon deletions predominating; and 1-4 bp insertions made up 5% of the characterized mutations. CrPic induced a statistically greater number of deletions and a statistically smaller number of base substitutions than have been measured in spontaneously generated mutants. These data confirm previous studies showing that CrPic is mutagenic, and support the contention that further study is needed to verify the safety of CrPic for human consumption.

  20. DNA and chromosome breaks induced by {sup 123}I-estrogen in CHO cells

    SciTech Connect

    Schwartz, J.L. |; Mustafi, R.; Hughes, A.; DeSombre, E.R.

    1997-07-01

    The effects of the Auger electron-emitting isotope I-123, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen positive Chinese hamster ovary (CHO-ER) cells. Exposure to the {sup 123}I-estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.2. The corresponding ratio with {sup 60}Co gamma rays was 15.6. The dose-response was biphasic suggesting that either receptor sites are saturated at high does, or that there is a nonrandom distribution of breaks induced by the {sup 123}I-estrogen. The {sup 123}I-estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1,000 disintegrations per cell. This corresponds to the mean lethal dose of {sup 123}I-estrogen for these cells suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that {sup 123}I-estrogen-induced chromosome breaks are rejoined. The F-ratio, the ratio of dicentrics to centric rings, was 5.8 {plus_minus} 1.7, which is similar to that seen with high LET radiations. Their results suggest that I-123 bound to estrogen is an efficient clastogenic agent, that the cytotoxic damage produced by I-123 bound to estrogen is very like high LET-induced damage, and the I-123 in the estrogen-receptor-DNA complex is probably in close proximity to the sugar-phosphate backbone of the DNA.

  1. Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

    PubMed

    Lin, Jian'er; Neo, Shu Hui; Ho, Steven C L; Yeo, Jessna H M; Wang, Tianhua; Zhang, Wei; Bi, Xuezhi; Chao, Sheng-Hao; Yang, Yuansheng

    2017-09-01

    Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Modulation of benzo(a)pyrene-induced toxicity and mutagenicity by conjugation enzymes in mammalian cells (CHO/HGPRT Assay)

    SciTech Connect

    Recio, L.

    1986-01-01

    The biotransformation of benzo(a)pyrene (BP) catalyzed by the mixed-function oxidase (MFO) system results in numerous metabolites which are cytotoxic and/or mutagenic to mammalian cells. However, these are conjugated with endogenous compounds such as glucuronic acid, sulfate and glutathione (GSH) resulting in detoxication. The effects of three conjugation enzyme systems, UDP-glucuronyltransferases (UDP-GT), sulfotransferases (ST) and glutathione S-transferases (GSHT) on BP-induced cytotoxicity and mutagenicity were studied using the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay. These studies were done by supplementing a rat-liver homogenate preparation containing MFO system cofactors. The relationship between BP 7,8-diol-9,10-epoxide (BPDE)-DNA adducts and mutagenicity in the CHO/HGPRT assay was also determined. The results of these studies are discussed. 149 refs., 11 figs., 11 tabs.

  3. Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap (laser tweezers)

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Liu, Yagang; Krasieva, Tatiana B.; Patrizio, Pasquale; Tadir, Yona; Sonek, Gregory J.; Berns, Michael W.; Tromberg, Bruce J.

    1995-05-01

    We describe fluorescence spectroscopy and imaging studies of optically trapped single Chinese hamster ovary (CHO) and motile human sperm cells. The NIR trapping beam was provided by a tunable, multimode continuous wave Ti:Sapphire laser. The beam was introduced into an inverted confocal laser scanning microscope. Fluorescence of cells in the single- beam gradient force optical trap was excited with a 488 nm microbeam (laser scanning microscopy) or with 365 nm radiation from a high- pressure mercury lamp. Modifications to NADH-attributed autofluorescence and Rhodamine- and Propidium Iodide-attributed xenofluorescence indicate a significant cell-damaging effect of 760 nm trapping beams. 760 nm effects produce a biological response comparable to UVA-induced oxidative stress and appear to be a consequence to two-photon absorption.

  4. Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

    PubMed

    Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

    1992-01-01

    We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the

  5. A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

    PubMed

    Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

    2015-09-01

    Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality.

  6. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

    PubMed

    Zhang, Ye; Stobbe, Per; Silvander, Christian Orrego; Chotteau, Véronique

    2015-11-10

    Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period. Copyright © 2015 The Authors. Published by Elsevier B.V. All

  7. Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

    PubMed Central

    Chowdhury, Ratul; Chowdhury, Anupam; Maranas, Costas D.

    2015-01-01

    Essentiality (ES) and Synthetic Lethality (SL) information identify combination of genes whose deletion inhibits cell growth. This information is important for both identifying drug targets for tumor and pathogenic bacteria suppression and for flagging and avoiding gene deletions that are non-viable in biotechnology. In this study, we performed a comprehensive ES and SL analysis of two important eukaryotic models (S. cerevisiae and CHO cells) using a bilevel optimization approach introduced earlier. Information gleaned from this study is used to propose specific model changes to remedy inconsistent with data model predictions. Even for the highly curated Yeast 7.11 model we identified 50 changes (metabolic and GPR) leading to the correct prediction of an additional 28% of essential genes and 36% of synthetic lethals along with a 53% reduction in the erroneous identification of essential genes. Due to the paucity of mutant growth phenotype data only 12 changes were made for the CHO 1.2 model leading to an additional correctly predicted 11 essential and eight non-essential genes. Overall, we find that CHO 1.2 was 76% less accurate than the Yeast 7.11 metabolic model in predicting essential genes. Based on this analysis, 14 (single and double deletion) maximally informative experiments are suggested to improve the CHO cell model by using information from a mouse metabolic model. This analysis demonstrates the importance of single and multiple knockout phenotypes in assessing and improving model reconstructions. The advent of techniques such as CRISPR opens the door for the global assessment of eukaryotic models. PMID:26426067

  8. CHO cell production and sequence improvement in the 13C6FR1 anti-Ebola antibody

    PubMed Central

    Pettit, Dean K.; Rogers, Richard S.; Arthur, Kelly; Brodsky, Yan; Clark, Rutilio H.; Crowell, Chris; Ennis, Jane; Gillespie, Alison; Gillespie, Ron; Livingston, Brittney; Nalbandian, Edith; Pace, Danielle; Smidt, Pauline; Pauly, Michael; Timmons, Ken; Trentalange, Michael; Whaley, Kevin J.; Zeitlin, Larry; Thomas, James N.

    2016-01-01

    ABSTRACT From March 2014 through February 2015, the Ebola virus spread rapidly in West Africa, resulting in almost 30,000 infections and approximately 10,000 deaths. With no approved therapeutic options available, an experimental antibody cocktail known as ZMapp™ was administered to patients on a limited compassionate-use basis. The supply of ZMapp™ was highly constrained at the time because it was in preclinical development and a novel production system (tobacco plants) was being used for manufacturing. To increase the production of ZMapp™ for an uncertain future demand, a consortium was formed in the fall of 2014 to quickly manufacture these anti-Ebola antibodies in Chinese hamster ovary (CHO) cells using bioreactors for production at a scale appropriate for thousands of doses. As a result of the efforts of this consortium, valuable lessons were learned about the processing of the antibodies in a CHO-based system. One of the ZMapp™ cocktail antibodies, known as c13C6FR1, had been sequence-optimized in the framework region for production in tobacco and engineered as a chimeric antibody. When transfected into CHO cells with the unaltered sequence, 13C6FR1 was difficult to process. This report describes efforts to produce 13C6FR1 and the parental murine hybridoma sequence, 13C6mu, in CHO cells, and provides evidence for the insertion of a highly conserved framework amino acid that improved the physical properties necessary for high-level expression and purification. Furthermore, it describes the technical and logistical lessons learned that may be beneficial in the event of a future Ebola virus or other pandemic viral outbreaks where mAbs are considered potential therapeutics. PMID:26761424

  9. Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

    PubMed

    Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

    2015-05-01

    CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

  10. Enhanced protein production by microRNA-30 family in CHO cells is mediated by the modulation of the ubiquitin pathway.

    PubMed

    Fischer, Simon; Mathias, Sven; Schaz, Simone; Emmerling, Verena Vanessa; Buck, Theresa; Kleemann, Michael; Hackl, Matthias; Grillari, Johannes; Aschrafi, Armaz; Handrick, René; Otte, Kerstin

    2015-10-20

    Functional genomics represent a valuable approach to improve culture performance of Chinese hamster ovary (CHO) cell lines for biopharmaceutical manufacturing. Recent advances in applied microRNA (miRNAs) research suggest that these small non-coding RNAs are critical for the regulation of cell phenotypes in CHO cells. However, the notion that individual miRNAs usually control the expression of hundreds of different genes makes miRNA target identification highly complex. We have recently reported that the entire miR-30 family enhances recombinant protein production in CHO cells. To better understand the pro-productive effects of this miRNA family, we set out to identify their downstream target genes in CHO cells. Computational target prediction combined with a comprehensive functional validation enabled the discovery of a set of twenty putative target genes for all productivity enhancing miR-30 family members. We demonstrate that all miR-30 isoforms contribute to the regulation of the ubiquitin pathway in CHO cells by directly targeting the ubiquitin E3 ligase S-phase kinase-associated protein 2 (Skp2). Finally, we provide several lines of evidence that miR-30-mediated modulation of the ubiquitin pathway may enhance recombinant protein expression in CHO cells. In summary, this study supports the importance of non-coding RNAs, especially of miRNAs, in the context of cell line engineering.

  11. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.

  12. Origin and evolution of binucleated cells and binucleated cells with micronuclei in cisplatin-treated CHO cultures.

    PubMed

    Rodilla, V

    1993-08-01

    It has recently been described that cisplatin is an agent able to induce binucleated cells (BC) in cultured CHO cells. Both the origin and the significance of those cells within a population are unknown although several hypothesis have been suggested such as blocking of cytokinesis or cell fusion. Using interval photography we have found that at least two mechanisms are involved in the production of BC. These cells can arise in a culture as a result of an incomplete process of cell division, i.e. karyokinesis with incomplete cytokinesis or as a result of the mitotic division of a pre-existent BC. The mitotic division of a BC can give rise to different types of daughter cells. These BC sometimes enter mitosis but fail to divide and as a consequence they remain BC. When the process of division is successful (in the vast majority of cases), the results that have been found are either two mononucleated cells or one mononucleated and one binucleated cell. The possible implications and significance of BC and BC with micronuclei in a given population are discussed.

  13. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    NASA Astrophysics Data System (ADS)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

  14. The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells.

    PubMed

    Rezaei, M; Zarkesh-Esfahani, S H; Gharagozloo, M

    2013-07-01

    Cell lines derived from mammalian are dominant systems for the production of recombinant therapeutic proteins because of their capacity for correct protein folding, assembly and post-translational modification. In the search of an efficient method for the production of a recombinant protein using animal cell culture, we investigated the effects of different treatment including fetal calf serum concentration, glycerol and culture temperature on a Chinese hamster ovary (CHO) cell line on the production of recombinant human growth hormone (rhGH) and recombinant Chinese hamster ovary (rCHO) viability. The GH production was assessed using ELISA and western blotting methods and cell viability was determined by flow cytometry. The production of recombinant protein increased by 2-fold when stimulatory chemical such as glycerol was added in two stages, first cells were cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol was added to achieve high specific productivity.. Moreover, glycerol addition increased cell viability. Low culture temperature (below 37°C) led to enhanced cellular productivity of the rhGH by 3-fold but decreased cell viability. These findings indicate that quite simple factors such as culture temperature and addition of simple chemicals may lead to the improvement of industrial process for the production of recombinant proteins such as rhGH.

  15. Induction of 8-hydroxy-2'-deoxyguanosine in CHO-K1 cells exposed to phenyl-hydroquinone, a metabolite of ortho-phenylphenol.

    PubMed

    Nakagawa, Y; Tayama, S

    1996-03-29

    The induction of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an index of oxidative DNA modification, was investigated in CHO-K1 cells exposed to phenyl-hydroquinone (PHQ), a major metabolite of ortho-phenylphenol (OPP), an antimicrobial. Addition of PHQ at a concentration of 50 microM to CHO cell suspensions (10(6) cells/ml) induced slight elevation of intracellular 8-OHdG levels. Pretreatment of CHO cells with 3-amino-1,2,4-triazole (AT, 20 mM) enhanced PHQ-induced 8-OHdG formation which was accompanied by cell death. Pretreatment of CHO-K1 cells with AT (20 mM) and deferoxamine (DeFe, 20 mM) inhibited the formation of 8-OHdG as well as cell death caused by PHQ. Neither AT nor DeFe affected cell viability or the formation of 8-OHdG in untreated CHO cells during the incubation period. The loss of cellular glutathione induced by the addition of PHQ alone was enhanced by the pretreatment of CHO cells with AT or AT plus DeFe. When PHQ was added to AT-pretreated cell suspensions, the concentration of PHQ decreased with time. This decrease was accompanied by the formation of phenyl-benzoquinone (PBQ). These results suggest that the reactive oxygen species derived from autoxidation of PHQ which converts to PBQ via phenyl-semiquinone elicit DNA damage in CHO cells, especially when the activity of cellular catalase is inhibited.

  16. Cytogenetic analyses of Azadirachtin reveal absence of genotoxicity but marked antiproliferative effects in human lymphocytes and CHO cells in vitro.

    PubMed

    Mosesso, Pasquale; Bohm, Lothar; Pepe, Gaetano; Fiore, Mario; Carpinelli, Alice; Gäde, Gerd; Nagini, Siddavaram; Ottavianelli, Alessandro; Degrassi, Francesca

    2012-09-18

    In this work we have examined the genotoxic potential of the bioinsecticide Azadirachtin A (AZA) and its influence on cell proliferation on human lymphocytes and Chinese Hamster ovary (CHO) cells. AZA genotoxicity was assessed by the analysis of chromosomal aberrations and sister chromatid exchanges (SCEs) in the absence and presence of rat liver S9 metabolism. Primary DNA damage was also investigated by means of the comet assay. The results obtained clearly indicate that AZA is not genotoxic in mammalian cells. On the other hand, AZA proved to interfere with cell cycle progression as shown by modulation of frequencies of first (M1) and second division (M2) metaphases detected by 5-Bromo-2'-deoxyuridine labeling. Accumulation of M1 metaphases were more pronounced in human lymphocytes. In the transformed CHO cell line, however, significant increases of multinucleated interphases and polyploid cells were observed at long treatment time. At higher dose-levels, the incidence of polyploidy was close to 100%. Identification of spindle structure and number of centrosomes by fluorescent immunostaining with α- and γ-tubulin antibodies revealed aberrant mitoses exhibiting multipolar spindles with several centrosomal signals. These findings suggest that AZA can act either through a stabilizing activity of microtubules or by inhibition of Aurora A, since both mechanisms are able to generate genetically unstable polyploid cells with multipolar spindles and multinucleated interphases. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

    PubMed

    Avgerinos, G C; Drapeau, D; Socolow, J S; Mao, J I; Hsiao, K; Broeze, R J

    1990-01-01

    We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

  18. Cytotoxicity Evaluation of Anatase and Rutile TiO2 Thin Films on CHO-K1 Cells in Vitro

    PubMed Central

    Cervantes, Blanca; López-Huerta, Francisco; Vega, Rosario; Hernández-Torres, Julián; García-González, Leandro; Salceda, Emilio; Herrera-May, Agustín L.; Soto, Enrique

    2016-01-01

    Cytotoxicity of titanium dioxide (TiO2) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO2 thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO2 films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO2 films’ thickness values fell within the nanometer range (290–310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO2 thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO2 thin films, the number of CHO-K1 cells on the control substrate and on all TiO2 thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO2 films annealed at 800 °C. These results indicate that TiO2 thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO2 thin films in biomedical science. PMID:28773740

  19. Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Kiseljak, Divor; Baldi, Lucia; Wurm, Florian M; Hacker, David L

    2015-01-01

    Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection.

  20. Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

    PubMed

    Johari, Yusuf B; Estes, Scott D; Alves, Christina S; Sinacore, Marty S; James, David C

    2015-12-01

    Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized

  1. Expression and regional assignment of Chinese hamster ESD and rRNA genes associated with translocations giving rise to chromosomes Z1 and Z6 in CHO cells.

    PubMed

    Stallings, R L; Adair, G M; Lin, J C; Siciliano, M J

    1984-01-01

    The Chinese hamster genes ADK, NP, ESD, PGM2, PEPS, PEPB, GLO, and GSR, all of which are on Chinese hamster chromosome 1, were assigned to CHO-LA chromosomes by analysis of the segregation of CHO isozymes and chromosomes from interspecific somatic cell hybrids made with CHO cells and mouse C11D cells. One allele of each of these eight loci remained linked on the normal chromosome 1 homolog. For seven loci, the other allele remained linked on chromosome Z1, but ESD was shown to have been translocated to chromosome Z6 (Chinese hamster chromosome 5q +). Ag-NOR staining of CHO chromosomes indicated that the (1;5) translocation was very likely reciprocal, since the Chinese hamster chromosome 5, which gave rise to the CHO Z6, lacks an NOR and the Z1 now has one. These data allowed regional assignment of ESD to the distal portion of Chinese hamster chromosome 1p and provided genetic evidence for the origin of CHO chromosomes Z1 and Z6 from Chinese hamster chromosomes 1 and 5. Induced electrophoretic shift mutations of ESD and positive Ag-NOR staining for the rRNA genes on the Z1 showed that the activities of the genes lying close to the translocation breakpoints were maintained.

  2. Carbamates: A study on genotoxic, cytotoxic, and apoptotic effects induced in Chinese hamster ovary (CHO-K1) cells.

    PubMed

    Soloneski, Sonia; Kujawski, Maciej; Scuto, Anna; Larramendy, Marcelo L

    2015-08-01

    In vitro effects of the carbamates pirimicarb and zineb and their formulations Aficida® (50% pirimicarb) and Azzurro® (70% zineb), respectively, were evaluated in Chinese hamster ovary (CHO-K1) cells. Whereas the cytokinesis-blocked micronucleus cytome assay was employed to test for genotoxicity, MTT, neutral red (NR), and apoptosis evaluation were used as tests for estimating cell viability and succinic dehydrogenase activity, respectively. Concentrations tested were 10-300 μg/ml for pirimicarb and Aficida®, and 1-50 μg/ml for zineb and Azzurro®. All compounds were able to increase the frequency of micronuclei. A marked reduction in the nuclear division index was observed after treatment with 5 μg/ml of zineb and Azzurro® and 10 μg/ml of Azzurro®. Alterations in the cellular morphology not allowing the recognition of binucleated cells exposed to 300 μg/ml pirimicarb and Aficida® as well as 10-50 μg/ml zineb and Azzurro®. All four compounds induced inhibition of both cell viability and succinic dehydrogenase activity and trigger apoptosis in CHO-K1 cells, at least when exposed for 24 h. The data herein demonstrate the genotoxic and cytotoxic effects exerted by these carbamates and reveal the potential risk factor of these pesticides, still extensively used worldwide, for both human health and the environment.

  3. Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

    PubMed Central

    Hanot, Camille C.; Choi, Young Suk; Anani, Tareq B.; Soundarrajan, Dharsan; David, Allan E.

    2015-01-01

    Superparamagnetic iron-oxide nanoparticles (SPIONs) show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells). We evaluated the effect of particle diameter (50 and 100 nm) and polyethylene glycol (PEG) chain length (2k, 5k and 20k Da) on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS). Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications. PMID:26729108

  4. Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement.

    PubMed

    Mosser, Mathilde; Kapel, Romain; Chevalot, Isabelle; Olmos, Eric; Marc, Ivan; Marc, Annie; Oriol, Eric

    2015-01-01

    Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

  5. Application of factorial design to accelerate identification of CHO growth factor requirements.

    PubMed

    Chun, Chung; Heineken, Katy; Szeto, Dongmei; Ryll, Thomas; Chamow, Steve; Chung, John D

    2003-01-01

    To accelerate recombinant CHO media and process development, we describe a simple approach to integrating multiple tasks associated with these processes including initial media design, serum-free adaptation, stability analysis and first generation scale-up. Factorial design techniques and normal probability chart representation of the results were first applied to identify potent parental CHO cell growth factors in a lean basal medium. These results were then applied to identify a suitable manufacturing medium from a panel of commercial and proprietary media formulations. When this approach was applied to recombinant CHO cell line, rapid adaptation of the cell line to an appropriate production medium occurred during culture expansion in the presence of the identified growth factor(s). This approach allows media component screening to be naturally integrated into the adaptation and scale-up processes since components that have little or no relative effect on cell proliferation are selected against as the "best" cultures are moved forward. The rapidity of the adaptation process allowed cell line stability studies to be initiated relatively early in the development process, thus providing preliminary stability information by the time the "outgrowing" culture could be scaled to 100-L reactors some 30 days after adaptation commenced. The application of full factorial design techniques allowed us to calculate the maximum number of interaction effects, the interpretation of which we believe can provide insights into growth factor biology.

  6. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    SciTech Connect

    Thompson, L.H.; Brookman, K.W.; Dillehay, L.E.; Mooney, C.L.; Carrano, A.V.

    1982-01-01

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m/sup 2/, each of the mutants showed < 10% of the incision rate of the parental AA8 cells. After 50 J/m/sup 2/, the rate in AA8 was similar to that at 6 J/m/sup 2/, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.

  7. Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells

    PubMed Central

    2011-01-01

    Introduction The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs) from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs. Methods An IDO expression plasmid was constructed and Chinese hamster ovary (CHO) cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control) CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127-]) subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression. Results IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127- T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P < 0.05) as well. The specific inhibitor of IDO,1-MT efficiently reversed enhancement of T cells apoptosis and amplification of Tregs in vitro. Increased expression of Foxp3, a key molecular marker of Tregs, was confirmed by RT-PCR, real-time RT-PCR and Western blot analysis at the same time. Conclusions These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression. PMID:21917155

  8. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells.

    PubMed

    Diepenbruck, Carolin; Klinger, Matthias; Urbig, Thomas; Baeuerle, Patrick; Neef, Rüdiger

    2013-06-01

    Selection of lead candidates in drug discovery is a complex and time-consuming process. Here, we describe an approach that allows prediction of the productivity and quality of recombinant proteins by stable producer cell clones with the help of transient transfection studies. This is exemplified for three distinct bispecific T cell engager (BiTE(®))-a new class of single-chain antibody-based therapeutics showing very promising results in the treatment of cancer. BiTE(®) titers of transiently transfected HEK cells showed a striking correlation with titers of selected stable CHO cell clones. Likewise, the percentage of the monomeric BiTE(®) fraction in cell culture supernatants correlated well between transiently expressing HEK and stably expressing CHO cell clones. This validates the use of transient transfection studies for the selection of biopharmaceutical lead candidates with desired pharmaceutical properties.

  9. Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells

    PubMed Central

    Skała, Ewa; Sitarek, Przemysław; Różalski, Marek; Krajewska, Urszula; Szemraj, Janusz; Wysokińska, Halina; Śliwiński, Tomasz

    2016-01-01

    Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress. PMID:27034736

  10. Analysis of Tubespins as a suitable scale-down model of bioreactors for high cell density CHO cell culture.

    PubMed

    Gomez, Natalia; Ambhaikar, Malhar; Zhang, Li; Huang, Chung-Jr; Barkhordarian, Hedieh; Lull, Jonathan; Gutierrez, Chris

    2017-03-01

    High cell density (HCD) culture increases recombinant protein productivity via higher biomass. Compared to traditional fed-batch cultures, HCD is achieved by increased nutrient availability and removal of undesired metabolic components via regular medium replenishment. HCD process development is usually performed in instrumented lab-scale bioreactors (BR) that require time and labor for setup and operation. To potentially minimize resources and cost during HCD experiments, we evaluated a 2-week 50-mL Tubespin (TS) simulated HCD process where daily medium exchanges mimic the medium replacement rate in BR. To best assess performance differences, we cultured 13 different CHO cell lines in simulated HCD as satellites from simultaneous BR, and compared growth, metabolism, productivity and product quality. Overall, viability, cell-specific productivity and metabolism in TS were comparable to BR, but TS cell growth and final titer were lower by 25 and 15% in average, respectively. Peak viable cell densities were lower in TS than BR as a potential consequence of lower pH, different medium exchange strategy and dissolved oxygen limitations. Product quality attributes highly dependent on intrinsic molecule or cell line characteristics (e.g., galactosylation, afucosylation, aggregation) were comparable in both scales. However, product quality attributes that can change extracellularly as a function of incubation time (e.g., deamidation, C-terminal lysine, fragmentation) were in general lower in TS because of shorter residence time than HCD BR. Our characterization results and two case studies show that TS-simulated HCD cultures can be effectively used as a simple scale-down model for relative comparisons among cell lines for growth or productivity (e.g., clone screening), and for investigating effects on protein galactosylation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:490-499, 2017. © 2017 American Institute of Chemical Engineers.

  11. Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

    PubMed Central

    de la Cueva, Ana; Ramírez de Molina, Ana; Álvarez-Ayerza, Néstor; Ramos, Ma Angeles; Cebrián, Arancha; del Pulgar, Teresa Gómez; Lacal, Juan Carlos

    2013-01-01

    Background Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. Methodology/Principal Findings ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. Conclusion/Significance Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors. PMID:23762272

  12. Chinese Hamster Ovary (CHO) Host Cell Engineering to Increase Sialylation of Recombinant Therapeutic Proteins by Modulating Sialyltransferase Expression

    PubMed Central

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R.; George, Henry J.; Brooks, Jeanne; Kayser, Kevin J.; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio-better” protein therapeutics and cell culture vaccine production. PMID:25641927

  13. Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression.

    PubMed

    Lin, Nan; Mascarenhas, Joaquina; Sealover, Natalie R; George, Henry J; Brooks, Jeanne; Kayser, Kevin J; Gau, Brian; Yasa, Isil; Azadi, Parastoo; Archer-Hartmann, Stephanie

    2015-01-01

    N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.

  14. Comparative radiation tolerance based on the induction of DNA double-strand breaks in tobacco BY-2 cells and CHO-K1 cells irradiated with gamma rays.

    PubMed

    Yokota, Yuichiro; Shikazono, Naoya; Tanaka, Atsushi; Hase, Yoshihiro; Funayama, Tomoo; Wada, Seiichi; Inoue, Masayoshi

    2005-05-01

    Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by gamma rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 +/- 0.1 DSBs Gbp(-1) Gy(-1)) was only one-third of that in CHO-K1 cells. Furthermore, the calculated number of DSBs per diploid cell irradiated with gamma rays at the mean lethal dose was five times greater in BY-2 cells (263 +/- 13) than in CHO-K1 cells. These results suggest that the radiation tolerance of BY-2 cells appears to be due not only to a lower induction of DNA damage but also to a more efficient repair of the induced DNA damage.

  15. Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines.

    PubMed

    Maszczak-Seneczko, Dorota; Olczak, Teresa; Wunderlich, Livius; Olczak, Mariusz

    2011-10-01

    Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCA(r) and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCA(r) mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCA(r) mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway.

  16. Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

    PubMed Central

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X.; Zheng, Lu; Pereira, Natasha A.; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  17. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

    PubMed

    Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

    2014-02-01

    The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

  19. Formation of recombinant triple-helical [alpha 1(IV)]2 alpha 2(IV) collagen molecules in CHO cells.

    PubMed

    Fukuda, K; Hori, H; Utani, A; Burbelo, P D; Yamada, Y

    1997-02-03

    Collagen IV molecules represent a major structural component of basement membranes providing a network of support for the supramolecular structure. Like other collagens, collagen IV forms a triple-helical molecule composed of three alpha chains. Six different alpha chains exist for collagen IV, although the most common isoform consists of two alpha 1(IV) and one alpha 2(IV) chain. To understand the molecular mechanism of triple-helical formation of collagen IV, we expressed recombinant alpha 1(IV) and alpha 2(IV) mouse collagen chains in Chinese hamster ovary (CHO) cells. An expression vector containing the full length cDNA for the mouse alpha 1(IV) chain was stably transfected into CHO cells and a cell line, A222, which expressed recombinant alpha 1(IV) chains was selected. These A222 cells were then infected with a retroviral expression vector containing the mouse alpha 2(IV) chain and a cell line, A222-A2, stably expressing both recombinant alpha 1(IV) and alpha 2(IV) chains was obtained. Immunoprecipitation of A222 cell lysates revealed a high level of alpha 1(IV) chain monomer, which was unable to form a homotrimer. Analysis of A222-A2 cell lysates revealed the presence of both monomeric alpha 2(IV) and alpha 1(IV) chains as well as a higher molecular weight collagen IV species. Second dimensional SDS-PAGE analysis demonstrated that the high molecular weight species was a heterotrimer consisting of two alpha 1(IV) and one alpha 2(IV) chain. This heterotrimer collagen IV species was pepsin-resistant indicating the formation of a stable triple-helical structure. Pulse-chase experiments showed that the monomer alpha 1(IV) chain was secreted, but at a much slower rate than the heterotrimer. Together these results demonstrate that the alpha 1(IV) chain is not capable of forming homotrimers and suggest that the coexpression with the alpha 2(IV) chain is necessary to form a triple-helical structure.

  20. Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20.

    PubMed

    Pieper, Lisa A; Strotbek, Michaela; Wenger, Till; Gamer, Martin; Olayioye, Monilola A; Hausser, Angelika

    2017-03-01

    Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins.

  1. Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It

    PubMed Central

    Kim, Dong-Ki; Choi, Hosoon; Nishida, Hidetaka; Oh, Joo Youn; Gregory, Carl; Lee, Ryang Hwa; Yu, Ji Min; Watanabe, Jun; An, Su Yeon; Bartosh, Thomas J.; Prockop, Darwin J.

    2016-01-01

    TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model. PMID:26793973

  2. Metabolomics profiling of extracellular metabolites in CHO-K1 cells cultured in different types of growth media.

    PubMed

    Mohmad-Saberi, Salfarina Ezrina; Hashim, Yumi Zuhanis Has-Yun; Mel, Maizirwan; Amid, Azura; Ahmad-Raus, Raha; Packeer-Mohamed, Vasila

    2013-08-01

    An efficient mammalian cell system for producing bioproducts should retain high cell viability and efficient use of energy sources rendering the need to understand the effects of various variables on the cell system. In this study, global metabolite (metabolomics) analysis approach was used to try and understand the relationships between types of media used, culture growth behavior and productivity. CHO-KI cells producing IGF-1 were obtained from ATCC and grown in T-flask (37 °C, 5 % CO2) until 70-80 % confluent in RPMI 1640 and Ham's F12, respectively. Samples were taken at 8-hourly intervals for routine cell counting, biochemical responses, insulin like growth factor-1 (IGF-1) protein concentration and global metabolite analysis (gas chromatography mass spectrometry, GCMS). Conditioned media from each time point were spun down before injection into GCMS. Data from GCMS were then transferred to SIMCA-P + Version 12 for chemometric evaluation using principal component analysis. The results showed that while routine analysis gave only subtle differences between the media, global metabolite analysis was able to clearly separate the culture based on growth media with growth phases as confounding factor. Different types of media also appeared to affect IGF-1 production. Asparagine was found to be indicative of healthiness of cells and production of high IGF-1. Meanwhile identification of ornithine and lysine in death phase was found to be associated with apoptosis and oversupplied nutrient respectively. Using the biomarkers revealed in the study, several bioprocessing strategies including medium improvement and in-time downstream processing can be potentially implemented to achieve efficient CHO culture system.

  3. miRNA profiling of high, low and non-producing CHO cells during biphasic fed-batch cultivation reveals process relevant targets for host cell engineering.

    PubMed

    Stiefel, Fabian; Fischer, Simon; Sczyrba, Alexander; Otte, Kerstin; Hesse, Friedemann

    2016-05-10

    Fed-batch cultivation of recombinant Chinese hamster ovary (CHO) cell lines is one of the most widely used production modes for commercial manufacturing of recombinant protein therapeutics. Furthermore, fed-batch cultivations are often conducted as biphasic processes where the culture temperature is decreased to maximize volumetric product yields. However, it remains to be elucidated which intracellular regulatory elements actually control the observed pro-productive phenotypes. Recently, several studies have revealed microRNAs (miRNAs) to be important molecular switches of cell phenotypes. In this study, we analyzed miRNA profiles of two different recombinant CHO cell lines (high and low producer), and compared them to a non-producing CHO DG44 host cell line during fed-batch cultivation at 37°C versus a temperature shift to 30°C. Taking advantage of next-generation sequencing combined with cluster, correlation and differential expression analyses, we could identify 89 different miRNAs, which were differentially expressed in the different cell lines and cultivation phases. Functional validation experiments using 19 validated target miRNAs confirmed that these miRNAs indeed induced changes in process relevant phenotypes. Furthermore, computational miRNA target prediction combined with functional clustering identified putative target genes and cellular pathways, which might be regulated by these miRNAs. This study systematically identified novel target miRNAs during different phases and conditions of a biphasic fed-batch production process and functionally evaluated their potential for host cell engineering.

  4. 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.

    PubMed

    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

  5. Effect of Lippia alba and Cymbopogon citratus essential oils on biofilms of Streptococcus mutans and cytotoxicity in CHO cells.

    PubMed

    Tofiño-Rivera, A; Ortega-Cuadros, M; Galvis-Pareja, D; Jiménez-Rios, H; Merini, L J; Martínez-Pabón, M C

    2016-12-24

    Caries is a public health problem, given that it prevails in 60 to 90% of the school-age global population. Multiple factors interact in its etiology, among them dental plaque is necessary to have lactic acid producing microorganisms like Streptococcus from he Mutans group. Existing prevention and treatment measures are not totally effective and generate adverse effects, which is why it is necessary to search for complementary strategies for their management. The study sought to evaluate the eradication capacity of Streptococcus mutans biofilms and the toxicity on eukaryotic cells of Lippia alba and Cymbopogon citratus essential oils. Essential oils were extracted from plant material through steam distillation and then its chemical composition was determined. The MBEC-high-throughput (MBEC-HTP) (Innovotech, Edmonton, Alberta, Canada) assay used to determine the eradication concentration of S. mutans ATCC 35668 strain biofilms. Cytotoxicity was evaluated on CHO cells through the MTT cell proliferation assay. The major components in both oils were Geraniol and Citral; in L. alba 18.9% and 15.9%, respectively, and in C. citratus 31.3% and 26.7%. The L. alba essential oils presented eradication activity against S. mutans biofilms of 95.8% in 0.01mg/dL concentration and C. citratus essential oils showed said eradication activity of 95.4% at 0.1, 0.01mg/dL concentrations and of 93.1% in the 0.001mg/dL concentration; none of the concentrations of both essential oils showed toxicity on CHO cells during 24h. The L. alba and C. citratus essential oils showed eradication activity against S. mutans biofilms and null cytotoxicity, evidencing the need to conduct further studies that can identify their active components and in order to guide a safe use in treating and preventing dental caries. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  6. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    SciTech Connect

    Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

    1983-01-01

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10/sup 6/ clonable cells. As with HGPRT/sup -/ mutants, XGPRT/sup -/ mutants can be selected in 6-thioguanine. AS52 (XGPRT/sup +/) and wild type CHO (HGPRT/sup +/) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT/sup -/ to HGPRT/sup -/ mutants induced per unit dose (J/m/sup 2/ for UV light and ..mu..g/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT/sup -/ AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment.

  7. An automated RNA-Seq analysis pipeline to identify and visualize differentially expressed genes and pathways in CHO cells.

    PubMed

    Chen, Chun; Le, Huong; Goudar, Chetan T

    2015-01-01

    Recent advances in RNA-Seq based comparative transcriptomics have opened up a unique opportunity to understand the mechanisms of different phenotypes in bioprocessing-related cell lines including Chinese hamster ovary (CHO) cells. However, simple and powerful tools are needed to translate large data sets into biologically relevant information that can be leveraged for genetic engineering and cell culture medium and process development. While tools exist to perform specific tasks associated with transcriptomics analysis, integrated end to end solutions that span the entire spectrum of raw data processing to visualization of gene expression changes on canonical pathways are rare. Additionally, these are not automated and require substantial user intervention. To address this gap, we have developed an automated RNA-Seq analysis pipeline in R which leverages the latest public domain statistical advances in transcriptomics data analysis. This pipeline reads RNA-Seq gene count data, identifies differentially expressed genes and differentially expressed pathways, and provides multiple intuitive visualizations as outputs. By using two publicly available CHO RNA-Seq datasets, we have demonstrated the utility of this pipeline. Subsequently, this pipeline was used to demonstrate transcriptomic similarity between laboratory- and pilot-scale bioreactors, helping make a case for the suitability of the lab-scale bioreactor as a scaled-down model. Automated end to end RNA-Seq data analysis approaches such as the one presented in this study will shorten the time required from acquiring sequencing data to biological interpretation of the results and can help accelerate the adoption of RNA-Seq analysis and thus mechanism-driven approaches for cell line and bioprocess optimization. © 2015 American Institute of Chemical Engineers.

  8. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells

    PubMed Central

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-01-01

    ABSTRACT Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR. PMID:28791335

  9. Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

    PubMed

    Park, Jong-Ju; Seong, Hun-Ki; Kim, Jeong-Soo; Munkhzaya, Byambaragchaa; Kang, Myung-Hwa; Min, Kwan-Sik

    2017-06-01

    Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn(56) of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

  10. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

    PubMed

    Montejo-López, Wilber; Rivera-Ramírez, Nayeli; Escamilla-Sánchez, Juan; García-Hernández, Ubaldo; Arias-Montaño, José-Antonio

    2016-09-01

    Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

  11. Mouse spinal cord neurons in serum-free culture media: suitability for patch clamp studies on chemical and electrical excitability.

    PubMed

    Salamanca, M C; Mathers, D A

    1987-01-01

    Methods were devised for the serum-free culture of spinal cord neurons derived from 12- to 13-day mouse embryos. Neurons exhibited good attachment if plated for 24 h on poly-d-lysine-coated dishes in the presence of serum. Cultures were subsequently fed with a serum-free medium consisting of minimum essential medium, Earle's salts and the N1 supplement, i.e. insulin, putrescine, transferrin, progesterone and selenium. After 3 weeks in vitro, growth and survival of neurons in this medium were comparable to results obtained using serum-supplemented medium. The presence of putrescine was not essential for the beneficial effects of N1, while insulin was required for long-term survival in serum-free media. Neurons maintained in serum-free media for 3 weeks retained aspects of electrical and chemical excitability characteristic of serum-grown cells.

  12. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: realizing the potential in bioreactors.

    PubMed

    Porter, Alison J; Dickson, Alan J; Racher, Andrew J

    2010-01-01

    Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake-flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake-flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS-CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake-flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered.

  13. Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

    PubMed Central

    Wilson, Paul F.; Hinz, John M.; Urbin, Salustra S.; Nham, Peter B.; Thompson, Larry H.

    2010-01-01

    The repair of DNA double-strand breaks (DSB) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and nonhomologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after γ-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300 cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction (~20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had ~2-fold higher chromatid-type CAs and a remarkable ~25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al. DNA Repair 4, 782–792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2. PMID:20434408

  14. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    PubMed

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  15. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    PubMed

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-03-06

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g. bispecific antibodies), cytokines or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent cell line development campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during cell line development in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial cell line development especially with regard to DTE proteins. This article is protected by copyright. All rights reserved.

  16. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    PubMed

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods.

  17. FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

    PubMed Central

    Li, Shengwei; Gao, Xiaoping; Peng, Rui; Zhang, Sheng; Fu, Wei

    2016-01-01

    A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells. PMID:27684722

  18. FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13).

    PubMed

    Li, Shengwei; Gao, Xiaoping; Peng, Rui; Zhang, Sheng; Fu, Wei; Zou, Fangdong

    A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells.

  19. CHO gene expression profiling in biopharmaceutical process analysis and design.

    PubMed

    Schaub, Jochen; Clemens, Christoph; Schorn, Peter; Hildebrandt, Tobias; Rust, Werner; Mennerich, Detlev; Kaufmann, Hitto; Schulz, Torsten W

    2010-02-01

    Increase in both productivity and product yields in biopharmaceutical process development with recombinant protein producing mammalian cells can be mainly attributed to the advancements in cell line development, media, and process optimization. Only recently, genome-scale technologies enable a system-level analysis to elucidate the complex biomolecular basis of protein production in mammalian cells promising an increased process understanding and the deduction of knowledge-based approaches for further process optimization. Here, the use of gene expression profiling for the analysis of a low titer (LT) and high titer (HT) fed batch process using the same IgG producing CHO cell line was investigated. We found that gene expression (i) significantly differed in HT versus LT process conditions due to differences in applied chemically defined, serum-free media, (ii) changed over the time course of the fed batch processes, and that (iii) both metabolic pathways and 14 biological functions such as cellular growth or cell death were affected. Furthermore, detailed analysis of metabolism in a standard process format revealed the potential use of transcriptomics for rational media design as is shown for the case of lipid metabolism where the product titer could be increased by about 20% based on a lipid modified basal medium. The results demonstrate that gene expression profiling can be an important tool for mammalian biopharmaceutical process analysis and optimization.

  20. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    SciTech Connect

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  1. Application of a cell-once-through perfusion strategy for production of recombinant antibody from rCHO cells in a Centritech Lab II centrifuge system.

    PubMed

    Kim, Byoung Jin; Chang, Ho Nam; Oh, Duk Jae

    2007-01-01

    Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

  2. Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells.

    PubMed

    Muluhngwi, Penn; Richardson, Kirsten; Napier, Joshua; Rouchka, Eric C; Mott, Justin L; Klinge, Carolyn M

    2017-03-15

    miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.

  3. Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations.

    PubMed

    Peñalber-Johnstone, Chariz; Ge, Xudong; Tran, Kevin; Selock, Nicholas; Sardesai, Neha; Gurramkonda, Chandrasekhar; Pilli, Manohar; Tolosa, Michael; Tolosa, Leah; Kostov, Yordan; Frey, Douglas D; Rao, Govind

    2017-07-01

    Cell-free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous-exchange cell-free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real-time. Mass transfer of important substrate and by-product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10-kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24-well plate high-throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next-generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478-1486. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization.

    PubMed

    Mozley, Olivia L; Thompson, Ben C; Fernandez-Martell, Alejandro; James, David C

    2014-01-01

    In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex

  5. Serum free light chain quantitative assays: Dilemma of a biomarker.

    PubMed

    Cigliana, Giovanni; Gulli, Francesca; Napodano, Cecilia; Pocino, Krizia; De Santis, Elena; Colacicco, Luigi; Cordone, Iole; Conti, Laura; Basile, Umberto

    2017-04-26

    Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory. We evaluated 300 serum samples from patients with B-cell disorders and compared the analytical performances of both assay. Each test was assayed on both testing platforms (Siemens Dade Behring BN II Nephelometer and SPAPLUS by The Binding Site). κ/λ ratios were determined and compared. Results were analyzed by Passing-Bablok and Bland-Altman plots to evaluate comparability of the two techniques and to determine bias. The reproducibility of both assays is acceptable, reaching minimum and desirable analytical goals derived from biological variability. However, values are not interchangeable between systems. This study shows that the two systems do not allow results to be transferred from one method to the other even if they display good agreement. Our study highlights the importance of elaborating an international standard for free light chains quantification in order to offer homogeneous results as well as guarantee harmonization of values among laboratories. Moreover, the assays should be validated in specific patient groups to determine that they are clinically fit for purpose. © 2017 Wiley Periodicals, Inc.

  6. A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells.

    PubMed

    Zalai, Dénes; Hevér, Helga; Lovász, Krisztina; Molnár, Dóra; Wechselberger, Patrick; Hofer, Alexandra; Párta, László; Putics, Ákos; Herwig, Christoph

    2016-08-01

    The integration of physiological knowledge into process control strategies is a cornerstone for the improvement of biopharmaceutical cell culture technologies. The present contribution investigates the applicability of specific productivity as a physiological control parameter in a cell culture process producing a monoclonal antibody (mAb) in CHO cells. In order to characterize cell physiology, the on-line oxygen uptake rate (OUR) was monitored and the time-resolved specific productivity was calculated as physiological parameters. This characterization enabled to identify the tight link between the deprivation of tyrosine and the decrease in cell respiration and in specific productivity. Subsequently, this link was used to control specific productivity by applying different feeding profiles. The maintenance of specific productivity at various levels enabled to identify a correlation between the rate of product formation and the relative abundance of high-mannose glycoforms. An increase in high mannose content was assumed to be the result of high specific productivity. Furthermore, the high mannose content as a function of cultivation pH and specific productivity was investigated in a design of experiment approach. This study demonstrated how physiological parameters could be used to understand interactions between process parameters, physiological parameters, and product quality attributes.

  7. In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

    PubMed Central

    Hasegawa, Haruki; Wendling, John; He, Feng; Trilisky, Egor; Stevenson, Riki; Franey, Heather; Kinderman, Francis; Li, Gary; Piedmonte, Deirdre Murphy; Osslund, Timothy; Shen, Min; Ketchem, Randal R.

    2011-01-01

    Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity. PMID:21464137

  8. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    SciTech Connect

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi; Fujita, Norihisa

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  9. A functional high-content miRNA screen identifies miR-30 family to boost recombinant protein production in CHO cells.

    PubMed

    Fischer, Simon; Buck, Theresa; Wagner, Andreas; Ehrhart, Carolin; Giancaterino, Julia; Mang, Samuel; Schad, Matthias; Mathias, Sven; Aschrafi, Armaz; Handrick, René; Otte, Kerstin

    2014-10-01

    The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO-SEAP cell line by means of a genome-wide high-content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell-specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR-30 family substantially improves bioprocess performance of CHO cells. Stable miR-30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR-30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

    PubMed

    El Naggar, Amel A; El-Naggar, Mostafa; Mokhamer, El-Hassan; Avad, Mona W

    2015-01-01

    The measurement of serum free light chain (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. The aim of the present work was to study the prognostic value of sFLC in multiple myeloma in relation to other serum biomarkers, response to therapy and survival. Forty five newly diagnosed patients with MM were included in the study. Patients were divided into responders and non-responders groups according to response to therapy. sFLC and serum Amyloid A (SAA) were measured by immunonephelometry. The non-responders group showed a statistically significant higher kappa/lambda or lambda/kappa ratio and higher β2 microglobulin level, but lower albumin level at presentation, as compared to the responders group (P < 0.001). However, no statistically significant difference was detected between the two groups regarding SA A or calcium levels. Comparison between sFLC ratio obtained before and after therapy revealed significant decrease after treatment in the responders group (P = 0.05). Survival was significantly inferior in patients with an FLC ratio of ≥ 2.6 or ≤ 0.56 compared with those with an FLC ratio that was between 0.56 and 2.6 (P = 0.002).

  11. A Murine Fibroblast Growth Factor (FGF) Receptor Expressed in CHO Cells is Activated by Basic FGF and Kaposi FGF

    NASA Astrophysics Data System (ADS)

    Mansukhani, Alka; Moscatelli, David; Talarico, Daniela; Levytska, Vera; Basilico, Claudio

    1990-06-01

    We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

  12. Pharmacological evaluation of alpha and beta human tachykinin NK(2) receptor splice variants expressed in CHO cells.

    PubMed

    Bellucci, Francesca; Meini, Stefania; Catalioto, Rose-Marie; Catalani, Claudio; Giuliani, Sandro; Quartara, Laura; Giolitti, Alessandro; Faiella, Angela; Rotondaro, Luigi; Candenas, Maria Luz; Pinto, Francisco M; Maggi, Carlo Alberto

    2004-09-24

    In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK(2) receptor splice variant named beta isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the alpha isoform. Moreover, neurokinin A was unable to inhibit the [(3)H]SR48968 binding to the beta isoform up to microM concentrations. In cells expressing the human tachykinin NK(2) receptor beta isoform, contrary to those expressing the alpha isoform, natural or selective tachykinin receptor agonists (1 microM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca(2+)](i). The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca(2+)](i) increase in cells expressing the alpha or beta isoform of the human tachykinin NK(2) receptor. The present data indicate that the human tachykinin NK(2) receptor beta isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK(2) receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands.

  13. Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

    PubMed Central

    Chen, D; Van Horn, D J; White, M F; Backer, J M

    1995-01-01

    Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK. PMID:7651388

  14. Approaches for recombinant human factor IX production in serum-free suspension cultures.

    PubMed

    do Amaral, Robson Luis Ferraz; de Sousa Bomfim, Aline; de Abreu-Neto, Mário Soares; Picanço-Castro, Virgínia; de Sousa Russo, Elisa Maria; Covas, Dimas Tadeu; Swiech, Kamilla

    2016-03-01

    To establish a serum-free suspension process for production of recombinant human factor IX (rhFIX) based on the human cell line HEK 293T by evaluating two approaches: (1) serum-free suspension adaptation of previously genetic modified cells (293T-FIX); and (2) genetic modification of cells already adapted to such conditions (293T/SF-FIX). After 10 months, 293T-FIX cells had become adapted to FreeStyle 293 serum-free medium (SFM) in Erlenmeyer flasks. After 48 and 72 h of culture, 2.1 µg rhFIX/ml and 3.3 µg rhFIX/ml were produced, respectively. However, no biological activity was detected. In the second approach, wild-type 293T cells were adapted to the same SFM (adaptation process took only 2 months) and then genetically modified for rhFIX production. After 48 h of culture, rhFIX reached 1.5 µg/ml with a biological activity of 0.2 IU/ml, while after 72 h, the production was 2.4 µg/ml with a biological activity of 0.3 IU/ml. The findings demonstrate that the best approach to establish an rhFIX production process in suspension SFM involves the genetic modification of cells already adapted to the final conditions. This approach is time saving and may better ensure the quality of the produced protein.

  15. Development and manufacturability assessment of chemically-defined medium for the production of protein therapeutics in CHO cells.

    PubMed

    Ling, Wai Lam W; Bai, Yunling; Cheng, Cheng; Padawer, Ishai; Wu, Changjian

    2015-01-01

    Advantages of using internally developed chemically-defined (CD) media for cell culture-based therapeutic protein production over commercial media include better raw material control and medium vendor options, and most importantly, flexibility for process development and subsequent optimization needed for therapeutic protein production. Through several rounds of design of experiment (DOE) screening, and medium component supplementation and optimization studies, we successfully developed a CD basal medium (CDM) for CHO cell culture. The internally prepared liquid CDM demonstrated comparable cell culture performance to that from a commercially available control medium. However, when the same CDM formulation was transferred to two major commercial medium suppliers for manufacturing, cell culture performance utilizing these newly prepared media was significantly reduced compared with the in-house prepared counterpart. An investigation was launched to assess whether key medium components were sensitive to large-scale preparation of the final bulk media by the vendors. Further work necessitated the reformulation of the original CDM formulation into a core medium that was suitable for large-scale media manufacturing. The modified preparation of the core medium with two separate supplements to generate the final CDM was able to recover the expected cell culture performance and monoclonal antibody (mAb) productivity. Confirmation of cell culture robustness in cell growth and production was corroborated in two additional mAb-expressing cell lines. This work demonstrates that a robust CD medium is not only one that performs during the development stage, but also one that must be reproducible by commercial media vendors. © 2015 American Institute of Chemical Engineers.

  16. Surface modification by argon plasma treatment improves antioxidant defense ability of CHO-k1 cells on titanium surfaces.

    PubMed

    de Queiroz, Jana Dara Freires; Leal, Angélica Maria de Sousa; Terada, Maysa; Agnez-Lima, Lucymara Fassarela; Costa, Isolda; Pinto, Nadja Cristhina de Souza; de Medeiros, Silvia Regina Batistuzzo

    2014-04-01

    Titanium is one of the most used materials in implants and changes in its surface can modify the cellular functional response to better implant fixation. An argon plasma treatment generates a surface with improved mechanical proprieties without modifying its chemical composition. Oxidative stress induced by biomaterials is considered one of the major causes of implant failure and studies in this field are fundamental to evaluate the biocompatibility of a new material. Therefore, in this work, induction of oxidative stress by titanium surfaces subjected to plasma treatment (PTTS) was evaluated. The viability of CHO-k1 cells was higher on PTTS discs. Cells grown on titanium surfaces are subjected to intracellular oxidative stress. Titanium discs subjected to the plasma treatment induced less oxidative stress than the untreated ones, which resulted in improved cellular survival. These were associated with improved cellular antioxidant response in Plasma Treated Titanium Surface (PTTS). Furthermore, a decrease in protein and DNA oxidative damage was observed on cells grown on the roughed surface when compared to the smooth one. In conclusion, our data suggest that the treatment of titanium with argon plasma may improve its biocompatible, thus improving its performance as implants or as a scaffold in tissue engineering. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Protective activity of Cynara scolymus L. leaf extract against chemically induced complex genomic alterations in CHO cells.

    PubMed

    Jacociunas, Laura Vicedo; de Andrade, Heloisa Helena Rodrigues; Lehmann, Mauricio; Pedersini, Larissa Wölfle; Ferraz, Alexandre de Barros Falcão; da Silva, Juliana; Dihl, Rafael Rodrigues

    2013-09-15

    Cynara scolymus L., popularly known as artichoke, has been widely used in traditional medicine as an herbal medicament for therapeutic purposes. The study aimed at assessing the protective activity of Cynara scolymus leaf extract (LE) against DNA lesions induced by the alkylating agent ethylmethnesulphonate (EMS) in Chinese hamster ovary cells (CHO). The ability of C. scolymus L. LE to modulate the mutagenicity of EMS was examined using the cytokinesis block micronucleus (CBMN) cytome assay in three antigenotoxic protocols, pre- post- and simultaneous treatments. In the pre-treatment, C. scolymus L. LE reduced the frequencies of MNi and NBUDs induced by EMS in the lower concentration. In contrast, at the highest concentration (5 mg/ml) artichoke enhanced the frequency of MNi, potentiating EMS genotoxicity. In the simultaneous treatment only the induction of MNi was repressed by the exposure of cells to C. scolymus L. LE. No modification in genotoxicity was observed in LE post-treatment. The results obtained in this study suggest that lower concentrations of artichoke prevent chemically induced genomic damage in mammalian cells. In this context, the protective activity of C. scolymus L. could be associated to its constitutive antioxidants compounds.

  18. Limitations in the process of transcription and translation inhibit recombinant human chorionic gonadotropin expression in CHO cells.

    PubMed

    Liu, Yang; Yi, Xiaoping; Zhuang, Yingping; Zhang, Siliang

    2015-06-20

    Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer with a α subunit and β subunit assembled together with disulfide bridges. This hormone plays an important role in the detection of ovulation induction and in the treatment of certain diseases that cause female infertility. The effects of transcription, subunit expression, assembling and secretion on recombinant hCG expression in CHO cells were studied using stable high-producing and low-producing cell lines generated by the FLP-In™ system. The results indicated that the mRNA and polypeptide levels of the β subunit were always higher than those of the α subunit. Further study confirmed that the differences were caused by the transcription rate rather than by mRNA stability. In the high-producing cell lines, there was obvious transcription level limitation of the α subunit in contrast to the β subunit. In addition, there was obvious limitation of the synthetic steps from mRNA to polypeptide for both the α subunit and the β subunit, especially the β subunit. Significant limitations of the assembly and secretion levels were not observed in this research. This study presents a research methodology for double subunit protein expression and provides valuable evidence for the enhancement of recombinant hCG productivity.

  19. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    PubMed Central

    Thoring, Lena; Wüstenhagen, Doreen A.; Borowiak, Maria; Stech, Marlitt; Sonnabend, Andrei; Kubick, Stefan

    2016-01-01

    Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called “difficult-to-express” proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of “difficult-to-express” proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called “cell-free” protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various “difficult-to-express” proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA. PMID:27684475

  20. Nucleases isolated from Chelidonium majus L. milky sap can induce apoptosis in human cervical carcinoma HeLa cells but not in Chinese Hamster Ovary CHO cells.

    PubMed

    Nawrot, Robert; Wołuń-Cholewa, Maria; Goździcka-Józefiak, Anna

    2008-01-01

    Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milk sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the dependence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 +/- 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 +/- 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflammatory reaction.

  1. Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells.

    PubMed

    Oda, T; Shinmura, N; Nishioka, Y; Komatsu, N; Hatakeyama, T; Muramatsu, T

    1999-04-01

    The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.

  2. Investigation of superparamagnetic (Fe3O4) nanoparticles and magnetic field exposures on CHO-K1 cell line

    NASA Astrophysics Data System (ADS)

    Coker, Zachary; Estlack, Larry; Hussain, Saber; Choi, Tae-Youl; Ibey, Bennett L.

    2016-03-01

    Rapid development in nanomaterial synthesis and functionalization has led to advanced studies in actuation and manipulation of cellular functions for biomedical applications. Often these actuation techniques employ externally applied magnetic fields to manipulate magnetic nanomaterials inside cell bodies in order to drive or trigger desired effects. While cellular interactions with low-frequency magnetic fields and nanoparticles have been extensively studied, the fundamental mechanisms behind these interactions remain poorly understood. Additionally, modern investigations on these concurrent exposure conditions have been limited in scope, and difficult to reproduce. This study presents an easily reproducible method of investigating the biological impact of concurrent magnetic field and nanoparticle exposure conditions using an in-vitro CHO-K1 cell line model, with the purpose of establishing grounds for in-depth fundamental studies of the mechanisms driving cellular-level interactions. Cells were cultured under various nanoparticle and magnetic field exposure conditions from 0 to 500 μg/ml nanoparticle concentrations, and DC, 50 Hz, or 100 Hz magnetic fields with 2.0 mT flux density. Cells were then observed by confocal fluorescence microscopy, and subject to biological assays to determine the effects of concurrent extreme-low frequency magnetic field and nanoparticle exposures on cellnanoparticle interactions, such as particle uptake and cell viability by MTT assay. Current results indicate little to no variation in effect on cell cultures based on magnetic field parameters alone; however, it is clear that deleterious synergistic effects of concurrent exposure conditions exist based on a significant decrease in cell viability when exposed to high concentrations of nanoparticles and concurrent magnetic field.

  3. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

    PubMed

    Balzac, F; Retta, S F; Albini, A; Melchiorri, A; Koteliansky, V E; Geuna, M; Silengo, L; Tarone, G

    1994-10-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

  4. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility

    PubMed Central

    1994-01-01

    The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125- kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with be