In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed an apoptosis-resistant, less lactate-producing dhfr(-) CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating LDH-A in a dhfr(-) CHO cell line. When the dhfr(-) CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were reduced to 21-65% and 37-78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant, less lactate-producing dhfr(-) CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing rCHO cells for producing therapeutic proteins.
BackgroundSerum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis.ResultsFrom a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM.ConclusionsThe use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.
Previously, overexpression of anti-apoptotic proteins, such as E1B-19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl-xL , a well-known anti-apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody-producing rCHO cell lines with regulated Bcl-xL overexpression (CS13*-0.02-off-Bcl-xL and CS13*-1.00-off-Bcl-xL ) were established using the Tet-off system. When cells were cultivated without Bcl-xL overexpression, the specific lactate production rate (qLac ) of CS13*-0.02-off-Bcl-xL and CS13*-1.00-off-Bcl-xL were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl-xL overexpression, in the absence of doxycycline, did not affect the qLac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose-6-phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl-xL overexpression either. Taken together, Bcl-xL overexpression showed no significant effect on the lactate metabolism of rCHO cells.
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