N-acetylcysteine and liberase improve success of hepatocyte isolation and viability of hepatocytes isolated from normal and diseased liver

BackgroundSuccessful hepatocyte isolation is crucial for development of cellular transplantation and biochemical research. Most researchers isolate hepatocytes from surplus donor tissue or normal tissue removed during resection of liver tumours. However, most tissue available for research is from explanted diseased liver and donor tissue rejected for transplant. We previously described our experience of hepatocyte isolation using liberase from such livers with a success rate of 51% and median viability of 40%. Liberase is a highly purified collagenase that has been shown to improve the viability of isolated porcine hepatocytes. N-acetylcysteine (NAC) has been shown to improve the viability of human hepatocytes isolated from steatotic donor tissue. The aim of this study was to determine the effect of both reagents in combination on the outcome of hepatocyte isolation from normal and diseased liver.MethodsHepatocytes were isolated from 30 consecutive liver specimens as previously described (old protocol). A further 30 consecutive liver specimens were perfused with buffer containing NAC and standard collagenase substituted by liberase (new protocol). Success was defined as maintenance of cell adhesion and morphology for 48 h and/or their successful use in laboratory studies. Mann-Whitney tests were used to compare results. Fisher's exact test was used for categorical data.FindingsBaseline factors were similar for both groups. The delay to tissue processing was slightly less in the new protocol group (median 2 h vs 4 h, p=0·007). The success rate improved from 40% (12/30) with the old protocol to 70% (21/30) with the new protocol (p=0·037), and the median viable cell yield increased from 7·3 × 10⁴ to 28·3 × 10⁴ cells per g tissue (p=0·003). After multivariable analysis adjusting for the difference in time delay, the success rate (p=0·014) and viable cell yield per g tissue (p=0·001) remained significantly improved.InterpretationNAC and liberase greatly improve the success of hepatocyte isolation and result in a significantly higher viable cell yield. Use of these agents may improve the availability of hepatocytes for transplantation as well as laboratory research.FundingUK Medical Research Council.