Schneider Y J, Lavoix A
Université Catholique de Louvain, Unité de Biochimie, Belgium.
J Immunol Methods. 1990 May 25;129(2):251-68. doi: 10.1016/0022-1759(90)90446-3.
We have recently shown that the semi-continuous cultivation of a mouse hybridoma line in spinner flasks, with a basal defined medium (BDM) devoid of serum and protein, increases the secretion of the immunoreactive monoclonal antibody (MAb) by a factor of ca. 2.4, compared to culture in flasks with serum-containing medium (Schneider, 1989). To further optimise MAb production, we have now investigated the composition of BDM and the mode of cultivation. Hybridoma cells were inoculated at 0.3 x 10(6) cells/ml in 200 ml of BDM containing 4, 6 or 8 mM glutamine; after 3-4 days (when the cell density reached greater than or equal to 10(6) cells/ml) 20, 40, 60 or 100% of the culture medium were replaced daily by fresh nutritive BDM with or without 33, 66 or 100% cell recycling. Daily, over a total period of 15-16 days, viable cells were counted and the concentration of the MAb, of glucose and glutamine (the main nutrients) and of lactate, alanine and ammonia (the main metabolites) were assayed. These experiments indicate that to provide optimal cell growth and MAb production: (i) the concentration of glutamine in the nutritive medium should be maintained at 8 mM, otherwise it falls on some days to 0 with a significant amount of cell death; at 25 mM, glucose concentration is not a limiting factor, whatever the culture conditions; (ii) with a semi-continuous mode of cultivation, a perfusion rate of 40%/day appears optimal; without cell recycling, a rate of 20% does not provide enough nutrients and/or does not remove enough metabolites, whereas a rate of 60% washes over the cells; with cell recycling, rates of 60 (moderately) or 100% (considerably) increase cellular metabolism without concurrent augmentation of MAb secretion; (iii) the recycling of the cells increases the mean cell densities and the rate of production of the MAb, as well as the rate of consumption of nutrients and of production of metabolites; recycling of 33-66% is optimal, since the total recycling progressively raises the number of dead cells and debris; (iv) there are maximal values for cell densities (ca. 2.5 X 10(6) cells/ml) and MAb production (ca. 26 micrograms/10(6) cells X day) as well as for nutrient consumption and metabolite production (except with a very high perfusion rate).(ABSTRACT TRUNCATED AT 400 WORDS)
我们最近发现，在无血清和蛋白质的基础限定培养基（BDM）中，于转瓶中对小鼠杂交瘤细胞系进行半连续培养，与在含血清培养基的培养瓶中培养相比，免疫反应性单克隆抗体（MAb）的分泌量增加了约2.4倍（施奈德，1989年）。为进一步优化单克隆抗体的生产，我们现在研究了BDM的组成和培养方式。将杂交瘤细胞以0.3×10⁶个细胞/毫升的密度接种于200毫升含4、6或8毫摩尔谷氨酰胺的BDM中；3 - 4天后（当细胞密度达到大于或等于10⁶个细胞/毫升时），每天用含或不含33%、66%或100%细胞回输的新鲜营养BDM替换20%、40%、60%或100%的培养基。在总共15 - 16天的时间里，每天对活细胞进行计数，并测定单克隆抗体、葡萄糖和谷氨酰胺（主要营养物质）以及乳酸、丙氨酸和氨（主要代谢产物）的浓度。这些实验表明，为实现最佳细胞生长和单克隆抗体生产：（i）营养培养基中谷氨酰胺的浓度应维持在8毫摩尔，否则某些日子里谷氨酰胺会降至0，导致大量细胞死亡；在25毫摩尔时，无论培养条件如何，葡萄糖浓度都不是限制因素；（ii）采用半连续培养模式时，每天40%的灌注率似乎最为适宜；不进行细胞回输时，20%的灌注率无法提供足够营养和/或无法清除足够代谢产物，而60%的灌注率会冲洗细胞；进行细胞回输时，60%（适度）或100%（大幅）的灌注率会增加细胞代谢，但不会同时增加单克隆抗体的分泌；（iii）细胞回输会增加平均细胞密度、单克隆抗体的产生速率以及营养物质的消耗速率和代谢产物的产生速率；33% - 66%的回输率最为适宜，因为完全回输会逐渐增加死细胞和碎片的数量；（iv）细胞密度（约2.5×10⁶个细胞/毫升）、单克隆抗体产量（约26微克/10⁶个细胞×天）以及营养物质消耗和代谢产物产生都存在最大值（极高灌注率情况除外）。（摘要截选至400字）
