Cryopreservation of Primarily Isolated Porcine Hepatocytes With UW Solution
Abstract:Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.
Document Type: Research Article
Affiliations: 1: *Division of Gastroenterology I, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 Japan 2: †Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan 3: ‡Medical Products Development Department, Kuraray Medical Co., 1621 Sakazu, Kurashiki, Okayama 710-8622, Japan 4: §Animal Center for Medical Research, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan 5: ¶Tissue Engineering Research Center, The National Institute of Advanced Industrial Science and Technology. 1-1-4 Higashi, Tsukubashi, Ibaragi 305-8562, Japan
Publication date: January 1, 2003
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