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

Abstract

This article describes a cell banking process for rBHK cell lines in 100-mL cryobags. As the use of larger volume cell banks requires greater cell numbers and longer preparation time, extensive characterization of key process parameters beyond the conventional ranges was performed to support a cGMP banking process. All experiments were conducted using two recombinant BHK21 cell lines, one of them cotransfected with Hsp70. The results show that the entire cell banking process for these BHK cell lines can be performed at room temperature. A DMSO exposure time up to 5 h either directly in a bioreactor or in shaker flasks did not result in any significant negative effect after cell thaw, when the cryocontainers were frozen immediately after filling. Extensive characterization did not indicate any significant apoptotic effects after thaw. However, the Hsp70 cotransfected cell line did show a slightly better protection from potential cryopreservation-induced apoptosis. Surprisingly, it was found that cells transferred into cryobags showed a low recovery rate after thaw if the incubation time exceeded 1.5 h before freezing. Additional experiments confirmed that the DMSO exposure time inside the cryocontainer in contrast to the DMSO exposure in a reactor or shaker flasks is much more critical. The cryobag cell banking process should therefore be performed within a 1(1/2)-2 h window; a banking process for vials should not exceed 2(1/2) h.

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.…”

The role of biopreservation in cell and gene therapy bioprocessing

“…This method of CRF has been demonstrated for both millilitre cryovials and in bags of large volumes (>100 mL) for both adult stem cells [11] and other mammalian cells [12,13]. It has also been successfully employed for the human fibroblast-derived dermal substitute Dermagraft (Advanced Biohealing, USA) [14] in addition to the hMSC product, Prochymal, which has received conditional approval in New Zealand and Canada, and is available in the United States under an Expanded Access Programme for treatment of acute GVHD in children [15,16].…”

Mammalian Expression System and Improvisation for High Production

“…Raman spectroscopy is used to analyze and maintain the consistency of the media components for large scale production [21]. Cryobag banking procedure was generated to shorten the time period from seed development to inoculums for production [22].…”