Premium
Cryopreservation in micro‐volumes: Impact upon caco‐2 colon adenocarcinoma cell proliferation and differentiation
Author(s) -
Malpique Rita,
KatsenGloba Alisa,
Carrondo Manuel J.T.,
Zimmermann Heiko,
Alves Paula M.
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21382
Subject(s) - cryoprotectant , cryopreservation , dimethyl sulfoxide , glycerol , chemistry , cell , microbiology and biotechnology , biophysics , biochemistry , biology , embryo , organic chemistry
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 µL 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 (Me 2 SO) 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 1 mL cryovials for cryopreservation of Caco‐2 cells using either Me 2 SO or glycerol. Compared to the manual process, the automatic addition of glycerol to the micro‐cryosubstrates allowed higher cell viabilities after thawing while with Me 2 SO no significant changes were observed. Me 2 SO 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 Me 2 SO 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. Biotechnol. Bioeng. 2007; 98: 155–166. © 2007 Wiley Periodicals, Inc.