Fluorinert, an oxygen carrier, improves cell culture performance in deep square 96‐well plates by facilitating oxygen transfer

Abstract

In bioprocess development, the 96-well plate format has been widely used for high-throughput screening of production cell line or culture conditions. However, suspension cell cultures in conventional 96-well plates often fail to reach high cell density under normal agitation presumably due to constraints in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well plate format, it often requires specialized instruments. In this report, we employed Fluorinert, a biologically inert perfluorocarbon, to improve oxygen transfer in 96-well plate and to enable the growth of a Chinese Hamster Ovary cell line expressing a recombinant monoclonal antibody. When different amounts of Fluorinert were added to the cell culture medium, a dose-dependent improvement in cell growth was observed in both conventional and deep square 96-well plates. When sufficient Fluorinert was present in the culture, the cell growth rate, the peak cell density, and recombinant protein production levels achieved in deep square 96-wells were comparable to cultures in ventilated shake flasks. Although Fluorinert is known to dissolve gases such as oxygen and CO(2), it does not dissolve nor extract medium components, such as glucose, lactate, or amino acids. We conclude that mixing Fluorinert with culture media is a suitable model for miniaturization of cell line development and process optimization. Proper cell growth and cellular productivity can be obtained with a standard shaker without the need for any additional aeration or vigorous agitation.

Establishment of a CFD‐based kLa model in microtiter plates to support CHO cell culture scale‐up during clone selection

“…A geometry with baffled walls was developed by Funke et al They investigated different baffled geometries and found an optimal design in the shape of a 6‐petaled flower regarding the oxygen transfer rates. Another method for improving the mass transfer is the addition of an oxygen transfer enhancing substance like Fluorinert, as performed by Meyer et al…”

Fluorinated methacrylamide chitosan hydrogels enhance collagen synthesis in wound healing through increased oxygen availability

“…PFCs are created by complete fluorination of hydrocarbons (replacement of hydrogen with fluorine), which generates a biologically non-reactive moiety [19]. The presence of a dense electron cloud, higher ionization potential, and greater electron affinity give rise to the highly hydrophobic and fluorophilic structure of PFCs [20].…”

Hypoxia and transforming growth factor‐beta1 pathway activation promote Chinese Hamster Ovary cell aggregation

“…Chemical oxygen carrier perfluorocarbons are organofluorine compounds and dissolve relatively high volumes of gases including oxygen, facilitating availability of oxygen, and being developed for blood substitutes (Cohn & Cushing, ). One of these compounds was tested in deep‐tube CHO cell cultures and found to improve culture performances because of improved oxygen supply (Meyer et al, ). To eliminate the confounding difference in physical dissociation and further investigate the role of hypoxia in promoting cell aggregation, eight (8) shake flasks of CHO cells were cultured at the low agitation speed (120 rpm) but half of them ( n = 4) were supported with a chemical oxygen carrier (Fluorinert FC‐770) at 20% v/v.…”