Large‐Scale, High‐density freezing of hybridomas and its application to high‐density culture

Abstract

Large-scale, high-density freezing of hybridomas was studied to apply frozen cells t o start high-density culture.We showed here that hybridomas can be frozen at 1.5 x lo8 cells/mL, without decrement in viability and proliferating activity. Blood transporting bags were used for large-scale freezing to store 25 mL of cell suspension with a cell density, 1.5 x lo8 cells/mL. The number of cells stored in a bag (3.0 x lo9 cells) was enough to start a high-density culture at a 10 times higher cell density (6.0 x lo6 cells/mL) than normal inoculation, and the cells proliferated to lo7 cells/mL within 2 days. These results indicate that the large-scale freezing method is useful for large-scale culture of mammalian cells.

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

“…This is in contrast to the findings of Ninomiya et al (1991) who reported that high-density seeding at 6 Â 10 6 cells/mL of hybridoma cells after cryopreservation shortened the lag period before IgM production started. However, the proliferating rate of the high-density seeded hybridoma cells was not improved, which shows that growth rate and productivity are not necessarily proportionally related to each other.…”

“…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. The exact mechanism of the protective properties of keeping the cells firmly packed during the freezing/thawing process is currently not known.…”

Quantitation of interaction of lipids with polymer surfaces in cell culture

“…This has been driven primarily by their ease of use and elimination of resource-intensive cleaning and sterilization procedures needed for more traditional glass or stainless steel vessels. Several groups have reported successful use of such disposable technologies for preparation of clinical materials under GMP conditions (Hami et al, 2004), for production of monoclonal antibodies (Ling et al, 2003) and cryostorage (Ninomiya et al, 1991). These disposable vessels typically consist of hydrophobic polymer films such as polyethylene (PE) and ethyl vinyl acetate (EVA).…”

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

“…The cryobags are designed to contain enough cells to inoculate the bioreactor directly without the necessity of any DMSO removal and the need of any additional small-scale culture vessels (Tflasks, spinners, roller bottles, etc.). The feasibility to use large-volume cryobags for cryopreservation in long-term storage conditions have been demonstrated in the past (Heidemann et al, 2002;Junker et al, 2002;Ninomiya et al, 1991;Regidor et al, 1998;Regidor et al, 1999). For example, Regidor et al 1998 reported stability data of umbilical cord blood preserved in blood transporting bags for more then 7 years.…”

“…Studies have demonstrated that the use of a large volume in combination with high density enables freezing and storage at about 100 times higher cell numbers than in the conventional case (2 mL cryovials). Cryobags successfully used in blood cell banking are used as a large-volume cryocontainer in place of conventional cryovials (Junker et al, 2002;Ninomiya et al, 1991;Regidor et al, 1999).…”