Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent.
Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 +/- 13 vs. 46.9 +/- 11%, P < 0.01) and plating efficiency (41.5 +/- 18 vs. 17.6 +/- 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation.
Katenz
Ekaterina
E
Department of General, Visceral, and Transplantation Surgery, Charité, Campus Virchow-Clinic, Universitätsmedizin Berlin, Germany. ekaterina.efimova@charite.de
Vondran
Florian Wolfgang Rudolf
FW
Schwartlander
Ruth
R
Pless
Gesine
G
Gong
Xiaobing
X
Cheng
Xiandong
X
Neuhaus
Peter
P
Sauer
Igor Maximilian
IM
D000418:Albumins / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D010865:Pilot Projects; D051381:Rats; D014199:Trehalose / Q000378:metabolism / Q000494:pharmacology*
D000418:Albumins; D003451:Cryoprotective Agents; D014199:Trehalose; D007770:L-Lactate Dehydrogenase; D001219:Aspartate Aminotransferases; D004121:Dimethyl Sulfoxide