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.
Mahler
Stephan
S
INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, 2 avenue Pr Léon Bernard, 35043 Rennes, France.
Desille
Mireille
M
Frémond
Benjamin
B
Chesné
Christophe
C
Guillouzo
André
A
Campion
Jean-Pierre
JP
Clément
Bruno
B
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