Cryopreservation of isolated primary rat hepatocytes: enhanced survival and long-term hepatospecific function.
OBJECTIVE
To investigate the long-term effect of cryopreservation on hepatocyte function, as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution, HypoThermosol (HTS), as the carrier solution.


BACKGROUND
Advances in the field of bioartificial liver support have led to an increasing demand for successful, efficient means of cryopreservation of hepatocytes.


METHODS
Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group), both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen, cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion, urea synthesis, deethylation of ethoxyresorufin (CYT P450 activity), and responsiveness to stimulation with interleukin-6 (IL-6) were performed.


RESULTS
Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo, in comparison with control (90%). Albumin secretion, urea synthesis and CYT P450 activity yielded 33%, 55%, and 59% in Media-cryo and 71%, 80%, and 88% in HTS-cryo, respectively, compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls.


CONCLUSIONS
This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability, long-term hepatospecific function, and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices.
Sosef
Meindert N
MN
Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Baust
John M
JM
Sugimachi
Keishi
K
Fowler
Alex
A
Tompkins
Ronald G
RG
Toner
Mehmet
M
D000818:Animals; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D019164:Liver, Artificial / Q000639:trends; D023421:Models, Animal; D019188:Organ Preservation Solutions / Q000494:pharmacology*; D051381:Rats; D011917:Rats, Inbred Lew
D019188:Organ Preservation Solutions; C494184:hypothermosol