Hepatocyte Isolation and Transplantation in the Pig

Abstract

Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of > 84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 10(9)) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.

Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination

“…After closing the abdomen, intravenous administration of 0.5 g/kg of D-Gal (Sigma, Saint Louis, MI) was conducted via the jugular route 24 h prior to cell transplantation. Both of the tubes were flushed with 1000 U of heparin (Aventis, Tokyo, Japan) to prevent blood clotting, capped, and then subcutaneously embedded for the following transplantation experiment, as previously reported [20]. Recipient pigs were randomly divided in the following groups: G1, single intraportal injection of reverted 16-T3 cells (1 · 10 9 cells) (n = 5); G2, single intraportal injection of primary isolated pig hepatocytes (1 · 10 9 cells, which were compatible to 5% mass of the whole of the pig liver) as a positive control (n = 3); and G3, intraportal injection of Ringer's Lactate solution as a negative control (n = 5).…”

“…Porcine hepatocytes were prepared from Landrace pigs (JA West of Okayama, Okayama, Japan), weighing 10 kg, by a four-step retrograde perfusion method using collagenase and dispase, as previously described [20]. The isolated hepatocytes with cell viability of >85% were used for transplant experiments.…”

Evaluation of a bioartificial liver based on a nonwoven fabric bioreactor with porcine hepatocytes in pigs

“…Hepatocytes were isolated from Chinese experimental miniature pigs using a modified four-step collagenase perfusion method as described elsewhere [31]. Firstly, excised liver lobes were perfused at 37 8C with 300 ml and then at 39 8C with 500 ml of perfusion buffer solution (PBS) composed of 9 g/L NaCl, 0.42 g/L KCl, 2.1 g/L NaHCO 3 , 0.9 g/L glucose and 4.78 g/L Hepes (Sigma-Aldrich Inc., USA) containing 0.37 g/L of EDTA (Gibco, USA).…”

MicroRNA-375 targets AEG-1 in hepatocellular carcinoma and suppresses liver cancer cell growth in vitro and in vivo

“…The characteristics of these patients are described in Supplementary Table S1. Normal PHHC were isolated using a modified four-step retrograde perfusion technique as reported previously (Maruyama et al, 2003;Meng et al, 2010). Cell morphologies of PHHC and hepatoma cell lines used in this study are shown in Supplementary Figure S1.…”