Spens Erika, Häggström Lena
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden.
Biotechnol Prog. 2005 Jan-Feb;21(1):87-95. doi: 10.1021/bp049822g.
A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.
已为NS0骨髓瘤细胞开发出一种化学成分明确、无蛋白且无动物成分的培养基，命名为RITM01。所用基础培养基是一种商业化的无血清、无蛋白杂交瘤培养基，并添加了磷脂酰胆碱、胆固醇、β-环糊精和柠檬酸铁。提高氨基酸浓度可显著改善细胞生长。一种组成型产生人IgG1抗体的NS0细胞系，在此培养基中达到了3×10⁶个细胞/毫升的峰值细胞密度。分批培养时抗体产量为195毫克/升，尽管细胞产量相同，但与标准补充血清的培养基相比增加了3倍。抗体产量的增加是由于生长阶段延长以及在低比增殖率下比抗体产生速率略有增加的结果。NS0骨髓瘤细胞系适应无蛋白条件大约需要3周时间，之后活力和细胞密度才会稳定。很可能发生了细胞存活和增殖所需的基因表达和表型行为变化。我们推测细胞自身产生的促有丝分裂因子参与了增殖的自分泌控制。为了研究此类因子的存在，研究了条件（用过的）培养基（CM）对细胞生长和增殖的影响。在细胞密度为2×10⁶个细胞/毫升时收获的10倍浓缩CM，即使仅以2.5%（v/v）供应，对增殖也有明显的积极影响。发现CM除了抗体外还含有大量细胞外蛋白质。在凝胶过滤柱上对CM进行分级分离，随后用各个级分补充新的NS0培养物，结果表明，与对照培养物相比，在20 - 25 kDa处洗脱的因子缩短了延迟期并增加了峰值细胞密度。鉴定参与增殖调控的自分泌因子可能会带来控制动物细胞过程中生长和产物形成的全新策略。
