Effects of hypothermia on the function, membrane integrity, and cytoskeletal structure of hepatocytes.
To increase the storage time of livers for transplantation, a better understanding of hypothermia-induced hepatocyte damage is necessary. To this end, we have characterized the effects of hypothermia on long-term function and cytoskeletal organization of hepatocytes cultured in the collagen sandwich configuration, which maintains the expression of liver-specific functions for several weeks. In these studies, cultured hepatocytes (maintained at 37 degrees C for 7 days) were exposed to 4 degrees C in Leibovitz-15 (L15), University of Wisconsin (UW) solution, or L15 supplemented with 2.5 g% polyethylene glycol (PEG) for various time periods followed by a return to normothermia. When L15 medium was used, the long-term albumin secretion rate of cultured hepatocytes was decreased by 50% after 4 h, and by 95% after 24 h of exposure to 4 degrees C. Amorphous precipitates of F-actin and fragmented short microtubules were also observed after 4 and 12 h of hypothermia, respectively. Similar results were obtained when hepatocytes were stored in UW solution. However, in L15 supplemented with PEG, no significant reduction in long-term albumin secretion rates and intact actin and microtubule morphology was observed even after 24 h of exposure to 4 degrees C. The membrane integrity and long-term albumin secretion of hepatocytes stored in the presence of PEG were decreased to approximately 50% only after 48 h of exposure to 4 degrees C. Thus, PEG may be a useful additive in preservation solutions for hepatocytes in hepatocyte-based liver support systems and for intact tissue as well.
Stefanovich
P
P
Surgical Services, Massachusetts General Hospital, Boston.
Ezzell
R M
RM
Sheehan
S J
SJ
Tompkins
R G
RG
Yarmush
M L
ML
Toner
M
M
D000199:Actins / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002470:Cell Survival; D002478:Cells, Cultured; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods; D003599:Cytoskeleton / Q000502:physiology / Q000648:ultrastructure; D008099:Liver / Q000166:cytology / Q000502:physiology; D008870:Microtubules / Q000648:ultrastructure; D051381:Rats; D013997:Time Factors
D000199:Actins; D000418:Albumins