A comparison of oxygenation methods fro high‐density perfusion culture of animal cells

Abstract

A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble ( approximately 5 mm in diameter), micron-sized bubble ( approximately 80 mum in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium.

Potential of cell retention techniques for large‐scale high‐density perfusion culture of suspended mammalian cells

“…Flow rates over 720 L и d −1 in the external loop were shown in some cases to be detrimental to cells (Zhang et al, 1993). High molecular weight products were reported to be retained by the membranes, inducing problems of product stability (Hiller et al, 1993).…”

“…Commercial modules (cellulose-ester fibers, 500-600 cm 2 , Microgon, Laguna Hills, CA, USA) were used by several investigators as retention devices for suspended cultures, either externally (Hiller et al, 1993;Zhang et al, 1993) or internally (Kyung et al, 1994), or even as microbioreactor for immobilized cultures (Tanase et al, 1997). The largest module available to date by Microgon for perfusion culture has a surface of 3.3 m 2 and is applicable to bioreactors ranging from 90-250 L. Another company proposes an original technology of Alternating Tangential Flow (ATF, Refine Technology Co., Edison, NJ, USA).…”

Mammalian cell retention devices for stirred perfusion bioreactors

“…Maiorella et al (1991) reported that cells were damaged at wall shear rates over 6000 s −1 , while Zhang et al (1993) observed a drop in culture viability at a shear rate of 1300 s −1 during perfusion using a hollow-fiber cartridge. Higher shear rates require higher inlet pressures and thus also increase the transmembrane pressure difference at the filter inlet.…”

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.…”