Dept. of Laboratory Medicine
20 Years of Experience in Isolation and Evaluation of Human Hepatocytes for Cell-Based Therapy
Roberto Gramignoli2, Kenneth Dorko1, Stephen C Strom2.
1Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, United States; 2Laboratory Medicine, Karolinska Institute, stockholm, Sweden
Background: Hepatocyte transplantation is gaining acceptance for the treatment of liver disease. Our laboratory has always been active in the translation of hepatocyte transplant technology from bench to clinic. Our 20 years’ experience in more than 2000 human livers allowed us to standardize reagents and procedures in accordance with Good Manufacturing Practice (GMP) and design rapid and sensitive functional assays to insure that cells for transplant function adequately.
Aim: A large number of human hepatocyte has been isolated to optimize a mechano-enzymatic procedure, and a large spectrum of primary cells have been analyzed to determine a normal range of activities to expect from isolated cells.
Methods: Different surgical procedures and enzymatic solutions have been tested starting in 1993 (i.e. Collagenase P, type IV or XI, Liberase®), since coming to a new collagenase-protease mixture (CIzyme®) specifically designed for the isolation of human hepatocytes. Data from viability/recovery and apoptosis to plating adhesion and hepatic function including basal and induced Cytochrome P450 (CYP) activities, phase II conjugation and ammonia metabolism were collected.
Results: Human hepatocytes have been isolated from 1315 post-natal and 706 fetal livers. Viability post-isolation has been going significantly improving from the early human cell preparations, used in the first clinical applications in the 90s (64±1%, mean±SEM) compared to the clinical hepatocyte transplants performed on 2012 (88±2%; p<0.0001). Cell recovery also significantly improved during the years. In the 90s we were able to isolate 1.5±0.2 million of viable hepatocytes per gram of tissue, while today we are collecting 8.4±1.8 million/gr. To ensure the quality of cell products, in some batch of cells we evaluated specific hepatic functions (i.e. CYP3A4). Considering that regulatory authorities require testing of quality and function of cells prior to transplantation, we adapted a large battery of test, including apoptosis and energy status evaluation, plating adhesion, ammonia metabolism and the phase I and II drug metabolism (CYP1A, 2C9, and 3A activities) providing information on metabolic status on cells immediately after isolation and their capacity to function on long-term.
Conclusions: Twenty years of experience in isolating hepatocytes leaded us to design and optimize a surgical/enzymatic GMP procedure to isolate human cells for clinical applications. We have been performed hepatocyte isolation not only on tissues rejected from organ donation, but also on liver resections performed for multiple clinical reasons, allowing us to enlarge the number of tissue to test enzymatic solutions and better understand the functional status. In addition, data collected on multiple hepatic functions worked as quality control and may form the basis for recipient’s need match criteria in hepatocytes transplant programs.
08:00 - 09:00
|Stem Cell Transplantation & Differentiation||Hepatocyte stem cells||Room 111-112|
15:30 - 17:00
|Stem Cells Bioengineering, and Preclinical Work||GMP Isolation and Quality Assesment on human Amnion Epithelial Cell for Clinical Therapies||Room 111-112|
11:00 - 12:30
|Hepatocyte Transplantation||20 Years of Experience in Isolation and Evaluation of Human Hepatocytes for Cell-Based Therapy||Room 111-112|
17:30 - 18:30
|Joint Poster Session||Efficient reprogramming of centenarian’s somatic cells, personalized geriatric medicine||Main Foyer 1 & 2|