Laboratório de Células Animais, Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, Av. Prof. Luciano Gualberto, Trav. 3, 380, Butantã, P.O. Box 61548, São Paulo, SP, 05508-900, Brazil,
Cytotechnology. 2014 Aug;66(4):605-17. doi: 10.1007/s10616-013-9612-0. Epub 2013 Jul 12.
This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.
本工作重点研究了不同搅拌通气系统和 pH 控制模式的两个搅拌罐生物反应器中溶解氧浓度 (DO) 对 BHK-21 细胞系（用于重组蛋白生产和病毒疫苗的宿主细胞）生长和代谢的影响。BHK-21 细胞系适应于单细胞悬浮培养，在 Celligen 中培养，不使用通气笼（旋转气体分布器）和 Bioflo 110，空气饱和度分别为 10%、30%和 50%（搅拌器用于从分布器环分散气体）。尽可能通过气体混合物将 pH 控制在 7.2。在其他运行中，在 30%和 50%（DO）的 Bioflo 110 中，细胞在 pH 通过 CO2 和 NaHCO3 溶液控制的条件下生长。通过酶法测定葡萄糖、乳酸、谷氨酰胺和铵的浓度。还测定了细胞浓度、大小和比耗氧量。当不使用 NaHCO3 溶液时，Celligen 和 Bioflo 110 的最佳 DO 分别为 10%和 50%空气饱和度。在这种条件下，最大细胞浓度高于 4×10(6)细胞/mL。在使用基础溶液添加的经典搅拌罐生物反应器（Bioflo 110）中，在 30%空气饱和度下进行的分批培养中，观察到最大细胞浓度增加了 36%。本工作中定义的最佳参数允许在两个具有不同搅拌通气系统的搅拌罐生物反应器中开发基于 BHK-21 细胞系的病毒疫苗、瞬时蛋白表达和基因治疗用病毒载体的生物工艺。
