Maintenance of Cold-Preserved Porcine Hepatocyte Function with UW Solution and Ascorbic Acid-2 Glucoside

Abstract

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 µg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 µg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 µg/ml) would be a useful cold preservation means for the development of cell therapies.Key words: Hepatocytes; University of Wisconsin solution; Ascorbic acid-2 glucoside; Cold preservation INTRODUCTIONcryopreservation method, and 3) establishment of a useful gene transfer technique for the isolated and/or cultured hepatocytes. Therefore, in the present study we inThe use of normal human hepatocytes in liver-targeted cell therapies is severely limited by worldwide donor vestigated an efficient procedure for isolating hepatocytes from a surgically resected segment of the pig liver. Deliver shortage. Considering the present status, it is critical to efficiently utilize such scarce donor livers for cell velopment of an effective preservation solution allows transportation of primarily isolated hepatocytes while therapies. What remains to be seen for the resolution are the following: 1) establishment of an excellent isolation maintaining adequate quality between one center where hepatocyte isolation and/or culture can be performed and procedure for functional and uniform hepatocyte preparation, 2) development of an effective cold storage and the other where such cells are actually utilized. Thus, we The resultant hepatocytes with more than 90% viabilCo., LTD., Tokyo, Japan) was intramuscularly used to ity assessed by a trypan blue exclusion test were subput the animals under sedation. An auricular vein was jected to a cold storage experiment. The following four secured to give droperidol 0.5 mg/kg (Sankyo Co.) and preservation s...

Hepatocyte Viability and Adenosine Triphosphate Content Decrease Linearly Over Time During Conventional Cold Storage of Rat Liver Grafts

“…For applications where primary hepatocyte isolation is required, such as bio-artificial livers, the ATP status can be a direct predictor for the expected viable cell yield per gram of liver tissue [24, 25], and the values determined in this work can provide an initial guideline for studies with human organs.…”

Characterization of the Radical-Scavenging Reaction of 2-O-Substituted Ascorbic Acid Derivatives, AA-2G, AA-2P, and AA-2S: A Kinetic and Stoichiometric Study

“…Third, AA-2G was reported to reduce oxidative stress in ischemic-reperfusion injury and has been studied as a useful means for organ transplantation. [33][34][35] We think that the radical-scavenging activity of AA-2G per se may contribute to the reduction of the oxidant stress because AA-2G appears to be metabolized only to a small extent to AA in the experiments carried out at Ϫ3°C or 4°C. Another question concerning the biological significance of AA derivatives arises from the fact that they did not scavenge the galvinoxyl radical at all, but other common antioxidants did (Fig.…”

Toward the survival and function of xenogeneic hepatocyte grafts

“…Indeed, it is widely believed that isolated cells can be stored at 4°C for 3–5 days without disturbing viability. Culture media, such as Williams‐E, Leibovitz media, and UW solution, have been recommended for this purpose 57, 58. We tested Williams‐E medium as well as UW solution; however, despite good viability after isolation, hepatocytes hypothermically preserved in Williams‐E medium showed a rapid decline in plating efficacy, upon transplantation did not survive in large numbers, and secreted far less albumin than those preserved in UW solution (data not shown).…”