Development and Application of Purified Tissue Dissociation Enzyme Mixtures for Human Hepatocyte Isolation
Abstract:Human hepatocyte transplantation is gaining acceptance for the treatment of liver diseases. However, the reagents used to isolate hepatocytes from liver tissue are not standardized and show lot-to-lot variability in enzyme activity and endotoxin contamination. For clinical application, highly purified reagents are preferable to crude digest preparations. A purified tissue dissociating enzyme (TDE) preparation (CIzymeTM purified enzymes) was developed based on the enzyme compositions found in a superior lot of collagenase previously used by our group for human hepatocyte isolation. The performance of this enzyme preparation was compared to collagenase type XI on 110 liver cases by assessing hepatocyte yield, viability, and seven other functional assays that included plating efficiency, basal and induced CYP450 activities, phase II conjugation activity, and ammonia metabolism. No statistically significant difference was observed between these TDEs when they were used to isolate hepatocytes from liver resections or organ donor tissue on 54 hepatocyte isolations with type XI enzyme and 56 isolations using CIzymeTM. These results show that a highly purified and defined TDE preparation can be formulated that provides excellent performance with respect to viability, yield, and functional activity of the isolated cells. In addition to reproducible formulation, these purified enzyme products have only 2‐3% of the endotoxin of crude enzyme preparations. These results show that purified enzymes such as CIzymeTM will be a safe and effective for the isolation of human hepatocytes for clinical transplants.
Document Type: Research Article
Affiliations: 1: Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 2: VitaCyte LLC, Indianapolis, IN, USA 3: Department of Sciences and Biomedical Technologies, Experimental Pathology Section, University of Cagliari, Cagliari, Italy 4: Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA, USA 5: Department of Clinical Science, Intervention and Technology (CLINTEC), Division of Transplantation Surgery, Liver Cell Lab, Karolinska University Hospital Huddinge, Stockholm, Sweden 6: Department of Transplant Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
Publication date: June 1, 2012
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