High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

Abstract

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10(8) cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…This is substantiated further by taking into account the reduction in q O 2 from 4.7 ± 0.6 pg/cell/h for NS/0 control cells to 2.9 ± 0.4 pg/cell/h for E1B-19K cells. Indeed, numerous studies focusing on lymphoid cell metabolism also present direct correlations between q O 2 and q S (Kyung et al, 1994;Ramirez and Mutharasan, 1990) and between q O 2 and specific growth rate (Banik and Heath, 1995;Boraston et al, 1984;Hiller et al, 1993;Ramirez and Mutharasan, 1990). …”

Effects of cell density and temperature on oxygen consumption rate for different mammalian cell lines

“…A feeding strategy based directly on OCR was illustrated for batch and fed-batch operation (Zhou and Hu, 1994). Similarly in a perfusion system, the feed rate to the bioreactor can be altered in response to variations in OCR (Kyung et al, 1994;Ozturk et al, 1995;Ozturk, 1996). Utilization of OCR for optimizing insect cell baculovirus systems was illustrated in the literature (Qualitz and Kwong, 1994).…”

“…In dynamic methods, the gas delivery is altered temporarily and the response in dissolved oxygen (DO) is analyzed for the determination of OCR (Kyung et al, 1994;Merten et al, 1986). In the continuous methods, the aeration system is not disturbed and the OCR can be calculated from mass balances using gas inlet and outlet composition.…”

Production of Recombinant Protein Using the HeLa S3-Vaccinia Virus Expression System: Bioreactor Perfusion and Effects of Post-Infection Temperature

“…Medium replacement via perfusion can dramatically boost cell density and yield. [20][21][22] The MOI also plays an important role in expression. Interestingly, previous work has shown that the optimal MOI was different for tissue flask and microcarrier cultures.…”

“…The hollow fiber system is relatively simple, requiring only a filter cartridge, tubing, and peristaltic pumps. Hollow fiber cartridges have been successfully used by other laboratories for perfusion of mammalian cell cultures, [20][21][22] and can handle large rates of exchange if sized properly. This report describes an evaluation of HeLa S3-vaccinia protein expression in hollow fiber perfusion culture.…”