Searles J A, Todd P, Kompala D S
Department of Chemical Engineering, University of Colorado, Boulder 80309-0424.
Biotechnol Prog. 1994 Mar-Apr;10(2):198-206. doi: 10.1021/bp00026a600.
The perfusion culture of suspended mammalian cells requires a cell retention device, the best of which will retain all viable cells and reject all nonviable cells and debris. The inclined settler is a passive, simple, inexpensive, and easy-to-maintain device that has been shown in the past to selectively remove single nonviable cells of hybridoma cultures. In this work, we have demonstrated the preferential return of viable recombinant Chinese hamster ovary (CHO) cells through the use of a three-port settler maintained at lower temperatures and vibrated to reduce cell attachment and enhance cell return to the bioreactor. The residence time of CHO cells in the cooled, vibrated settler was determined by flow-cytometric discrimination of tracer recombinant CHO cells. Cells returning to the bioreactor through the underflow had an average residence time of 1.46 h in the settler. During perfusion cultures with cell densities above 10(6) cells/mL, cells seen to be stalled within the settler were easily dislodged by periodic air bubbling using a simple back-flushing procedure in which headspace gas was brought through the settler underflow port. The resuspended cells were returned to the bioreactor within an average of 32 min after bubbling. This study demonstrates that inclined sedimentation technology can be utilized to selectively recycle viable recombinant CHO cells with only a short retention time in an inclined settler.
悬浮哺乳动物细胞的灌注培养需要一个细胞截留装置，其中最好的装置能截留所有活细胞并排斥所有死细胞和碎片。倾斜沉降器是一种被动、简单、廉价且易于维护的装置，过去已证明它能选择性地去除杂交瘤培养物中的单个死细胞。在这项工作中，我们通过使用一个保持在较低温度并振动以减少细胞附着并增强细胞返回生物反应器的三通沉降器，证明了活的重组中国仓鼠卵巢（CHO）细胞的优先回流。通过流式细胞术对示踪重组CHO细胞进行鉴别，确定了CHO细胞在冷却、振动沉降器中的停留时间。通过底流返回生物反应器的细胞在沉降器中的平均停留时间为1.46小时。在细胞密度高于10(6) 个细胞/毫升的灌注培养过程中，通过使用简单的反冲洗程序，即让顶空气体通过沉降器底流端口，周期性鼓泡可轻松使滞留在沉降器内的细胞脱落。鼓泡后，重新悬浮的细胞平均在32分钟内返回生物反应器。这项研究表明，倾斜沉降技术可用于在倾斜沉降器中仅以较短的保留时间选择性地回收活的重组CHO细胞。
