RESUMO
Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins.
Assuntos
Apoptose , Proliferação de Células , Regulação da Expressão Gênica , L-Lactato Desidrogenase/genética , Animais , Técnicas de Cultura Celular por Lotes , Células CHO , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Sobrevivência Celular , Clonagem Molecular , Cricetinae , Cricetulus , Meios de Cultura/química , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Potencial da Membrana Mitocondrial , Ácido Pirúvico/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lactato de Sódio/químicaRESUMO
In laboratory scale therapeutical protein production, cell clumps form typically in shake flasks, which hinders cell growth and decreases protein yield. To minimize clumps during the culture of Chinese hamster ovary cells, we employed the combination of two reagents, dextran sulfate (DS) and recombinant trypsin (r-trypsin). Our results showed that both DS and r-trypsin could diminish cell aggregation when adding them respectively, but clumps were still noticed obviously. In order to further mitigate cell agglomerate, a combination of 1.2 g/L DS and 8.0 mg/L r-trypsin was employed and no clumps were found under the bright field microscope. Strikingly, the highest viable cell density of combination group was increased from 5.12 × 10(6) to 7.13 × 10(6) cells/mL, while the integral of viable cells concentration was raised from 35.13 × 10(6) to 60.87 × 10(6) cells·days/mL, and the culture period was prolonged by 4 days. In addition, the antibody integrity was maintained in the combination group compared with that of the control.