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.

Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators

“…As iron chelator, deferoxamine and/or the new lipophilic hydroxamic acid derivative LK 614 appear applicable. In recent years, organ preservation solutions have -in addition to their use in classical organ transplantation -increasingly been used for the cold storage of cells and tissues [42][43][44][45][46][47][48][49]. It is likely, with the current efforts in the diverse fields of cellular therapies, as well as the advances in tissue engineering, that this use of preservation solutions will further increase in the future.…”

Serum-free cryopreservation of porcine hepatocytes

“…Evidence also exists that cryopreserved macroencapsulated pig hepatocytes survive after transplantation of the cells in minitubes into rats, as demonstrated after explantation of the minitubes and assessment of the functional properties of the cells (Sarkis et al 2001). Only one study is available describing the transplantation of freshly thawed porcine hepatocytes into galactosamine-treated rats; this improved the survival of the animals for several days compared with nontransplanted animals, indicating the survival and proper functioning of the transplanted cells (Kunieda et al 2003). However, because this approach used a xenogeneic animal model without immunosuppression, conclusions cannot be drawn regarding the survival of cryopreserved porcine hepatocytes in a syngeneic system.…”

“…To our knowledge, only two reports are available to date that provide serum-free hepatocyte cryopreservation protocols for the use of murine (Canaple et al 2001) and porcine (Kunieda et al 2003) hepatocytes. The report by Canaple et al (2001) describes the functional maintenance of cryopreserved hepatocytes by alginate encapsulation for up to 4 days in culture.…”

“…The report by Canaple et al (2001) describes the functional maintenance of cryopreserved hepatocytes by alginate encapsulation for up to 4 days in culture. Kunieda et al (2003) used UW solution for cryopreservation and obtained cell recoveries for porcine hepatocytes of about 50%; however, fetal bovine serum was included during the thawing process. The results obtained here with porcine hepatocytes may not be directly transferable to the situation with human hepatocytes.…”

“…In view of European Union guidelines regulating good manufacturing practice (GMP) for cells and tissues (Commission Directive 2003/94/EC) and safety guidelines for donation, procurement, testing, processing, storage and distribution of human tissues and cells (Commission of the European Union, COM (2002) 319/F), the development of protocols for the serum-free cryopreservation of hepatocytes for long-term storage is mandatory. A number of protocols for the cryopreservation of hepatocytes from different sources in the presence of serum are currently in use: freshly isolated primary hepatocytes (Alexandre et al 2002;Dandri et al 2001;de Sousa et al 1999;Hengstler et al 2000a;Hewitt et al 2001;Jamal et al 2000;Kunieda et al 2003;Silva et al 1999;Steinberg et al 1999), short-term cultured hepatocytes (Chen et al 2001;Koebe et al 1996) and alginate-encapsulated hepatocytes (Canaple et al 2001;Guyomard et al 1996;Rialland et al 2000;Vian et al 2002). According to these protocols, cryopreserved hepatocytes may be used in metabolic and toxicological studies or as a source for the configuration of bioartificial liver systems with potential downstream applications.…”