Proliferation of rat small hepatocytes after long-term cryopreservation.
OBJECTIVE
The demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established.


METHODS
Colonies of small hepatocytes were collected and then maintained at -80 degrees C for more than 6 months. Albumin secretion and mRNA expression of thawed cells were measured by enzyme linked immunosorbent assay and Northern blotting, respectively, and the expression of hepatic functions was examined by immunoblotting. The ultrastructure of cryopreserved cells was also examined.


RESULTS
About 60% of the cryopreserved colonies attached on dishes and then proliferated. The average area of small hepatocyte colonies was about 7.5 times larger at day 15 than at day 1. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed carbamoyl phosphate synthetase I and tryptophan 2,3-dioxygenase.


CONCLUSIONS
Small hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation.
Ikeda
Shinichiro
S
Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chu-Ku, S-1, W-17, Sapporo, Japan.
Mitaka
Toshihiro
T
Harada
Keisuke
K
Sugimoto
Shinichi
S
Hirata
Kohichi
K
Mochizuki
Yohichi
Y
D000418:Albumins / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D048429:Cell Size; D002478:Cells, Cultured; D015925:Cryopreservation; D005109:Extracellular Matrix; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D020675:Peroxisomes / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley
D000418:Albumins