Optimal conditions for freezing CHO‐S and HEK293‐EBNA cell lines: Influence of Me₂SO, freeze density, and PEI‐mediated transfection on revitalization and growth of cells, and expression of recombinant protein

Abstract

To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

“…Cell cryopreservation in amounts large enough to directly inoculate a bioreactor has been described some years ago for hybridoma cells . Cryopreservation in bags is widely used for blood cell storage and has been studied for suspension of CHO and BHK cells stored from shake flask or bioreactor culture at 20 × 10 6 cells/mL or transient expression of HEK293 cells in suspension . Over the last decade, cell banking of large cell amounts, achieved by high cell density and large stored volume, has had an increasing interest in the biopharmaceutical industry.…”

“…21 Cryopreservation in bags is widely used for blood cell storage and has been studied for suspension of CHO and BHK cells stored from shake flask or bioreactor culture at 20 3 10 6 cells/mL 22,23 or transient expression of HEK293 cells in suspension. 24 Over the last decade, cell banking of large cell amounts, achieved by high cell density and large stored volume, has had an increasing interest in the biopharmaceutical industry. It has the obvious advantage of a gain of 2-3 weeks of time and labor compared to the classical approach of small-volume cryovials at low cell density.…”

GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process

“…These include retention of good functionality, sterility, and scale up: for example, estimates for a bioartificial liver indicate that cryopreservation of 2 L of alginate-encapsulated cell spheroids (ELS) are required. 2 Cryopreservation in bags of large volumes ( > 100 mL) of adult stem cells 3 and of mammalian tissue culture cells [4][5][6] using conventional liquid nitrogen-based controlled rate freezers (CRFs) is established. However, the use of liquid nitrogen is problematic in a GMP environment; following manufacture, liquid nitrogen contains very low levels of contaminants, but during transport and storage it can become contaminated by ice, inanimate debris, and viable microorganisms [7][8][9] that can transfer to the local environment.…”

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

“…Historically, fetal bovine serum concentrations for cryopreservation mixtures are usually defined in a 10-95 % (v/v) range (Kloth et al 2010;Kleman et al 2008). This change Fig.…”