Hypothermic storage and cryopreservation of hepatocytes: the protective effect of alginate gel against cell damages.
Hepatocyte-based therapy has been proposed as an alternative to organ transplantation in the treatment of liver disorders. In the clinical context, a major issue is the constant supply of quality assurance-controlled hepatocytes, thereby requiring their cold storage in good conditions. We have analyzed the protective effects of alginate entrapment of rat hepatocytes after either 24 or 48 h of hypothermic storage or cryopreservation on the cell viability, cell yield, both mitochondrial and other cytoplasmic functional activities, and apoptosis. Decrease in viability, as evaluated by the MTT inclusion test, was 4% and 13% (24 h at 4 degrees C), 15% and 33% (48 h at 4 degrees C), and 9% and 19% (liquid nitrogen) for entrapped and free suspended hepatocytes, respectively. Viable cell yields were 86 +/- 8% and 51 +/- 6% for cryopreserved entrapped and free suspended hepatocytes, respectively. The mitochondrial (MTS assay), 7-ethoxyresorufin O-deethylase (EROD), and glutathione-S-transferase (GST) activities were better preserved in entrapped than in free suspended hepatocytes. Both hypothermic storage and cryopreservation were found to induce early caspase-3-like activities, being always much lower in entrapped hepatocytes, particularly after cryopreservation (98.4 +/- 42.4 vs. 6.4 +/- 4.0 fluorescence arbitrary units/hours/microg protein). Thus, cold-induced apoptosis in hepatocytes can be significantly reduced following their entrapment within alginate gel beads and this is associated with an improvement of both their viability and function.
INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, 2 avenue Pr Léon Bernard, 35043 Rennes, France.
Stephan
S
Mahler
Mireille
M
Desille
Benjamin
B
Frémond
Christophe
C
Chesné
André
A
Guillouzo
Jean-Pierre
JP
Campion
Bruno
B
Clément
D000464:Alginates / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D017690:Cell Transplantation / Q000379:methods* / Q000639:trends; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005782:Gels / Q000494:pharmacology / Q000627:therapeutic use; D020723:Glucuronic Acid / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology / Q000637:transplantation*; D006603:Hexuronic Acids / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008107:Liver Diseases / Q000628:therapy; D016031:Liver Transplantation / Q000379:methods* / Q000639:trends; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D051381:Rats
D000464:Alginates; D003451:Cryoprotective Agents; D005782:Gels; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D007770:L-Lactate Dehydrogenase; D005982:Glutathione Transferase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases