Pierson B A, Europa A F, Hu W S, Miller J S
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455, USA.
J Hematother. 1996 Oct;5(5):475-83. doi: 10.1089/scd.1.1996.5.475.
Large-scale ex vivo expansion of human natural killer (NK) cells for adoptive immunotherapy requires assurance of good manufacturing practices. However, maximal expansion of NK is also desired to facilitate clinical trials with large numbers of IL-2-activated NK (ANK). A closed-system stirred-tank bioreactor is amenable to computer control of culture variables, thereby reducing the risk of contamination. We demonstrate that NK cultured in 250-ml spinner flasks expand 2.5-fold more than NK cultured in stationary tissue culture wells. We further show that during 33 days of culture, it is feasible to control the pH between 7.0 and 7.2 and the dissolved oxygen concentration at 40% of air saturation via direct on-line computer control in a 750-ml stirred-tank bioreactor. On-line measurement of optical density by a laser turbidity sensor, as a measure of cell concentration, correlated well with actual cell count data. NK expansion in the 750-ml bioreactor was 7-fold greater than in stationary tissue culture controls and 3-fold greater than in spinner flask controls. Consumption rates of glucose and oxygen and the production rate of lactate were measured and will be used to develop a nutrient feeding strategy to convert the batch reactor experiment into closed-system fed-batch or continuous flow modes. Computer-controlled stirred-tank bioreactors may facilitate clinical trials with high-purity ANK populations for adoptive immunotherapy.
为进行过继性免疫治疗而对人自然杀伤（NK）细胞进行大规模体外扩增需要确保良好生产规范。然而，也希望NK能实现最大程度的扩增，以推动针对大量白细胞介素2激活的NK（ANK）细胞的临床试验。封闭式搅拌罐生物反应器适合对培养变量进行计算机控制，从而降低污染风险。我们证明，在250毫升转瓶中培养的NK细胞比在静止组织培养孔中培养的NK细胞扩增了2.5倍。我们进一步表明，在33天的培养过程中，通过在750毫升搅拌罐生物反应器中进行直接在线计算机控制，将pH值控制在7.0至7.2之间，并将溶解氧浓度控制在空气饱和度的40%是可行的。通过激光浊度传感器在线测量光密度作为细胞浓度的指标，与实际细胞计数数据相关性良好。在750毫升生物反应器中NK细胞的扩增比静止组织培养对照高7倍，比转瓶对照高3倍。测量了葡萄糖和氧气的消耗速率以及乳酸的产生速率，并将用于制定营养物进料策略，将分批反应器实验转换为封闭式补料分批或连续流动模式。计算机控制的搅拌罐生物反应器可能有助于针对过继性免疫治疗的高纯度ANK细胞群体进行临床试验。
