Isolation and Cryopreservation of Rat Hepatocytes

Hepatocytes were isolated from 250 to 350 g Sprague Dawley rats (Charles River, Harlow, UK), by in situ collagenase perfusion of the liver as previously described [27 (link)]. The cell suspension was centrifuged at 50×g for 5 min at 4 °C to isolate the hepatocytes. Cell viability was determined using trypan blue and always reached at least 65%. Cells were cryopreserved at 1 × 10⁷ cells/ml in UW solution with 5% glucose and 10% DMSO and thawed at time of transplant [28 (link)]. To allow cell tracking, the transplanted cells were stained with 60 μM CM-DiL, a lipophilic carbocyanine cell tracker dye, excitation 553 nm, emission 570 nm (Thermo Fisher Scientific, Paisley, UK). Cells were incubated with the CM-DiL dye at 37 °C for 5 min and then at 4 °C for 15 min. Unbound dye was washed off by centrifugation at 50×g for 5 min. In addition, eGFP rat hepatocytes were isolated from Lewis-Tg (CAG-eGFP) Ysrrc rats and are known as HepaCur™. The cells were kindly donated by Yecuris, Portland, OR, USA.