Primary exploration of conditions for hypothermic preservation of rat hepatocyte spheroids

Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium (SFM). Spheroids were then maintained in rocking culture at 37°C(control condition), or cold stored at 4°Cfor 24 or 48 hours in four different cold storage solutions: SFM alone; SFM+1mmol/L deferoxamine (Def); SFM+1|xmol/L cyclosporin A (CsA); and SFM+1mmol/L Def+1|xmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def was significantly higher than that stored in SFM or SFM+CsA(P<0.05). The production of albumin was in low level in SFM, SFM+Def, SFM+CsA and SFM+Def+CsA groups, and there was no difference between them (P>0.05). Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases.