Hepatocyte isolation and transplantation in syngenic rats

Refinement of techniques to isolate viable hepatocytes began in the late 1960's. It was established that perfusion of the intact liver as opposed to incubation of liver slices or chopped tissue increased the yield of cells. The present study aims to establish a simple, two-step, collagenase digestion method for hepatocyte isolation. A single inbred Fisher rat was used for hepatocyte isolation. The liver was perfused in-situ with perfusion buffer containing ethylene glycol bis N, N1, tetra acetic acid (EGTA), followed by the collagenase buffer. The liver was excised and gently minced. The tissue was resuspended in the collagenase buffer to complete dissociation. The cell suspension obtained was washed, centrifuged and filtered to complete the isolation procedure. The trypan blue exclusion test showed 80-85% cell viability. The isolated cells were transplanted into the splenic parenchyma of syngenic rats. Survival of the transplanted hepatocytes was confirmed by histological examination at the end of 90 days. This two step technique of in-situ liver perfusion gives a high yield of viable hepatocytes which show long term survival after transplantation.