Serum-free cryopreservation of five mammalian cell lines in either a pelleted or suspended state

Abstract

Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K), we found that the serum-free alternative preserves nearly as efficiently as the serumcontaining preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

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).…”

Characterization of cell‐banking parameters for the cryopreservation of mammalian cell lines in 100‐mL cryobags

“…To reduce or to eliminate the damage to cells, standard cryopreservation protocols recommend the addition of DMSO to the cell suspension at low temperatures (generally 4 C) and also to minimize of the DMSO exposure time before freezing. As a common supplement of cell culture media, bovine serum was used for several decades as a cryoprotectant in freezing media for mammalian cell lines (Corsini et al, 2004). Currently, in commercial manufacturing processes, the use of serum or any other animal-derived components has several disadvantages.…”

Rapid cryopreservation of five mammalian and one mosquito cell line at −80°C while attached to flasks in a serum free cryopreservative

“…Previous work with multi-well plates has shown that a variety of cell lines can remain viable during storage while attached to substratum of multi-well plates (1-3), thus eliminating time consuming steps. In addition, our recent experiments with serum-free cryopreservatives have shown that a phosphate-buffered saline (PBS)/10% dimethyl sulfoxide (DMSO) solution performs nearly as well as a serum-containing preservative (4). Together, these findings suggested the possibility of cryostoring cells with a serum-free preservative while attached to a 25cm 2 tissue culture flask.…”

“…With this in mind, we have extended our initial studies (1, 4) in three ways. First, we show that cells stored in a frozen state while attached to substratum of 25 cm 2 culture flasks remain viable for 30-180 days at -80°C.…”