Cryopreservation and gel collagen culture of porcine hepatocytes.
OBJECTIVE
To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation.


METHODS
Hepatocytes, isolated from Chinese experimental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel.


RESULTS
Viability of 150 mL/L DMSO group thawed hepatocytes was (83+/-4)%, but after purification, its viability was (90+/-5)%, attachment efficiency was (86+/-7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatocytes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days' culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups.


CONCLUSIONS
Storage in liquid nitrogen can long keep hepatocytes' activities, the concentration of 150 mL/L DMSO is fit for porcine hepatocytes' cryopreservation. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.
Liu
Hong-Ling
HL
Research Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
Wang
Ying-Jie
YJ
Guo
Hai-Tao
HT
Wang
Yu-Ming
YM
Liu
Jun
J
Yu
Yue-Cheng
YC
D000818:Animals; D002448:Cell Adhesion; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen; D015925:Cryopreservation; D005782:Gels; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D013552:Swine; D013556:Swine, Miniature
D005782:Gels; D003094:Collagen