Cryopreservation and gel collagen culture of porcine hepatocytes

AIM:To study the method of cryopreserving porcinehepatocytes and gel collagen culture measure after itscryopreservation.METHODS:Hepatocytes,isolated from Chinese experimentalsuckling mini-pigs by two-step perfusion with collagenaseusing an extra corporeal perfusion apparatus,werecryopreserved with 50 mL/L to 200 mL/L DMSO in liquidnitrogen for 4 mo,then thawed and seeded in 1 or between2 layers of gel collagen.The expression of porcine albuminmessage RNA,cellular morphology and content of aspartateaminotransferase (AST) and urea nitrogen (UN) wereexamined during culture in gel.RESULTS:Viability of 150 mL/L DMSO group thawedhepatocytes was (83±4)%,but after purification,its viabilitywas (90±5)%,attachment efficiency was (86±7)%,theviability of thawed hepatocytes was near to fresh cells.Whenthe thawed hepatocytes were cultivated in gel collagen withculture medium adding epidermal growth factor,thehepatocytes grew in various administrative levels in mixedcollagen gel,and bunchy in the sandwich configurationcultures.For up to 10 days’ culture,the typical cellularmorphological characteristics of cultivated hepatocytes couldbe observed.The leakage of AST was lower during culturein gel than that in common culture.At the same time,theUN synthesized by cells cultivated in mixed gel collagen washigher than that in other groups.CONCLUSION:Storage in liquid nitrogen can long keephepatocytes’ activities,the concentration of 150 mL/L DMSOis fit for porcine hepatocytes’ cryopreservation.Thawedhepatocytes can be cultivated with collagenous matrix,whichprovides an environment that more closely resembles thatin vivo and maintain the expression of certain liver-specificfunction of hepatocytes.