Cryopreservation of myeloma cells, hybridoma cells, and lymphocytes with different cryoprotectants

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

“…In this study, a Me 2 SO concentration exceeding 10% had a pronounced negative effect on the recovery of cells upon thawing and suggests that HEK293-EBNA and CHO-S cells are relatively sensitive to Me 2 SO during the process of freezing. Our results show that the generally accepted standard protocol for cryopreservation of mammalian cell lines using 10% Me 2 SO (Corsini et al, 2004;Doyle et al, 1999;Freshney, 1994;Merten et al, 1995;Neronov et al, 1992;Sasaki et al, 2005) is not specific. In line with our results, careful studies of survival rate following freezing in media without serum using different Me 2 SO concentrations have shown that cryopreserved human fibroblasts and keratinocytes tolerate 5% better than 10% Me 2 SO (Baust et al, 2002).…”

“…Recommendations for freezing mammalian cells describe the use of 10% Me 2 SO and a freezing cell density between 1 Â 10 6 and 10 Â 10 6 cells/mL (Corsini et al, 2004;Neronov et al, 1992). The DOE was designed around these recommendations and Me 2 SO concentrations in the range of 4-15%, freezing densities between 1 Â 10 6 and 50 Â 10 6 cells/mL and seeding densities between 0.3 Â 10 6 and 2 Â 10 6 cells/mL were tested (see Table I).…”

“…Interestingly, the identified optimum freezing density of 20-50 Â 10 6 cells/mL is quite high and differs from the common standard procedure to cryopreserve cells that involves freezing with a density of 1 Â 10 6 cells/mL to 10 Â 10 6 cells/mL and a Me 2 SO concentration of around 10% (Fieder et al, 2005;Freshney, 1994;Merten et al, 1995;Neronov et al, 1992). High-density freezing (from 20 to 40 Â 10 6 to 150 Â 10 6 cells/mL) in the presence of 10% Me 2 SO have also been performed successfully with BHK, CHO-S (Heidemann et al, 2002) and hybridoma (Ninomiya et al, 1991) cell lines.…”

“…However, Me 2 SO may also cause intracellular stress due to its hydrophobic interactions with cellular proteins. The generally accepted standard procedure for cryopreservation of mammalian cells involves low cooling rates of 1-38C/min prior to long-term storage at À1508C and fast thawing in a 378C water bath (Day and McLellan, 1995;Doyle et al, 1999;Neronov et al, 1992). During thawing, extra-and possibly intracellular recrystallization may occur if the warming process is too slow and as a consequence the cell walls will be challenged by mechanical stress (Mazur et al, 1972).…”

Stability of Recombinant Protein Production in the GS‐NS0 Expression System Is Unaffected by Cryopreservation

“…There are various reports in the literature concerning establishment of, and variations to, the standard procedure for cyropreservation. For instance, the effects of different cryoprotectants, different concentrations of cryoprotectants, and different rates of cooling have all been characterized (Ashwood-Smith, 1964;Birkeland, 1976;Faraci et al, 1976;Thorpe et al, 1976;Brock and Baartz, 1980;Neronov et al, 1992). Other studies have examined the effects of temperature changes on the cryopreservation of Chinese hamster ovary cells (Kaeffer et al, 1994) and hybridoma cells (Wewetzer and Dilmaghani, 2001).…”