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Cryopreservation in micro-volumes: Impact upon caco-2 colon adenocarcinoma cell proliferation and differentiation

 
: Malpique, R.; Katsen-Globa, A.; Carrondo, M.J.T.; Zimmermann, H.; Alves, P.M.

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Biotechnology & bioengineering 98 (2007), Nr.1, S.155-166
ISSN: 0006-3592
ISSN: 1097-0290
Englisch
Zeitschriftenaufsatz
Fraunhofer IBMT ()

Abstract
Recent advances in cell-based therapies require new approaches for cell cryopreservation, capable of dealing with large number of samples and providing specific conditions for each cell type. Reduction of sample volume from the commonly used 1 ml, to 25 mu L, in 30-well micro-cryosubstrates improves cryopreservation by allowing automation, data handling and access to individual wells without thawing the whole cryosubstrate. This system was evaluated for the storage of Caco-2 colon adenocarcinoma cells, which differentiate spontaneously after long-term culture. The impact of the cryosample small volume upon post-thawing membrane integrity of the cells and their capacity to proliferate and differentiate was studied. Two different cryoprotectants commonly employed, dimethyl sulfoxide (Me2SO) and glycerol, were evaluated as well as the possibility of decreasing their concentration from the 10% concentration, usually used, down to 3% (v/v). The process automation by pipette robotic addition of the cryoprotectant to the micro-cryosubstrates was also evaluated. The micro-cryosubstrates have proven to be at least as efficient as typical I mL cryovials for cryopreservation of Caco-2 cells using either Me2SO or glycerol. Compared to the manual process, the automatic addition of glycerol to the inicro-cryosubstrates allowed higher cell viabilities after thawing while with Me2SO no significant changes were observed. Me2SO has shown to be more effective than glycerol in maintaining high post-thaw cell membrane integrity, either in micro-cryosubstrates or cryovials, for any of the concentrations tested. The ability of Me2SO in maintaining high cell membrane integrity post-thawing was confirmed by long-term (up to 22 days) proliferation and differentiation studies performed with cells cultured immediately after thawing.

: http://publica.fraunhofer.de/dokumente/N-63147.html