Methods for obtaining high viable cell density in mammalian cell culture
A technology of cell culture and living cells, applied in the direction of cell culture active agents, animal cells, cell culture medium, etc., can solve the problem of not investigating the influence of the metabolic state of anti-apoptotic genes
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[0046] In the following example, CHO cell lines overexpressing anti-apoptotic genes were analyzed for maximum viable cell density, lifespan, caspase-3 activation and mitochondrial membrane potential (MMP) in shake flask cultures. Additionally, nutrient consumption and metabolite production in these cell lines were compared to control cell lines to determine whether the expression of anti-apoptotic genes had any effect on nutrient consumption and metabolite accumulation in mammalian cell culture systems.
[0047] Materials and methods :
[0048] cell culture :
[0049] CHOK1SV cell line (Lonza Biologics, Slough, UK) (designated as control cell line C1013A) was cultured in CD-CHO medium (Catalog No. 10743-011, Invitrogen, Carlsbad, CA). In some cases, another animal protein-free medium (defined as high glucose medium) containing glucose at various concentrations (including 60 mM) was used. Fetal bovine serum was purchased from Hyclone Labs, Logan, UT (Catalogue # SH30071....
example 1
[0074] Generation and characterization of apoptotic resistant cell lines
[0075] Table 1 above shows a list of the cell lines used in this study, the expression plasmids transfected in each case to generate the cell lines and the selection agents used to isolate the transfectomas. The listed cell lines represent multiple clones generated from each transfection. The name of each cell line is derived from the anti-apoptotic gene that was transfected into the host cell line. For example, EAX197 is a cell line transfected with E1B-19K (E), Aven (A) and XIAPΔ (X). The term "anti-apoptotic" is used to refer to cell lines that have been transfected with one or more of these anti-apoptotic genes.
[0076] All apoptosis-resistant cell lines used in this study were characterized by Western blot, caspase 3 / 7 activity, and mitochondrial membrane potential (data not shown). Single transfectant E64 or double transfectants EA63, EA112, EA167 and EA190 expressed levels of E1B-91K above ...
example 2
[0083] Effect of E1B-19K on Viable Cell Density of CHOK1SV
[0084] In Figure 4, a double transfectant overexpressing E1B-19K together with Aven (EA167), a triple transfectant overexpressing E1B-19K together with Aven and XIAPΔ (EAX197), and a transfectant overexpressing E1B-19K only (E64) compared to a control (host) cell line. The highest VCD of the control cell line reached 7.7×10 6 cells / ml, the highest VCD of E64 (only expressing E1B-19K) was 8.9×10 6 cells / ml, while the highest VCD of EA167 was 1.4×10 7 cells / ml, the highest VCD of EAX197 was more than 1.5×10 7 cells / ml, an increase of more than 190% compared to the control cell line. EA167 and EAX197 showed a drastic decrease in viability at days 8 and 9 post-inoculation, presumably due to nutrient depletion, whereas control and E64 cell lines showed a slower decrease in viability ( Figure 4B ). Figure 4C The net effect on IVCC is shown. The IVCC of the control cell line was 4.9 × 10 7 cells / day / ml, the IVCC...
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