Xenogeneic hepatocyte transplantation might offer an unobtrusive alternative to whole liver allotransplantation. Having previously found that the immune response to such grafts can be controlled by immunosuppression, we sought approaches to collection and delivery that would optimize survival and function after transplantation. Porcine hepatocytes were isolated by a 2-step collagenase technique and then: 1) used immediately; 2) stored in University of Wisconsin (UW) solution at 4 degrees C; 3) cultured in supplemented Williams E medium; or 4) cryopreserved in UW solution with 10% dimethyl sulfoxide (DMSO). The fate and function of the hepatocytes was determined after they were injected into the spleens of immunodeficient mice. Freshly isolated hepatocytes had better viability (92.2 +/- 1.9%) than hepatocytes cultured for 24 hours (78.4 +/- 6.3%), hypothermically preserved in UW solution for 24 hours (85.8 +/- 3.1%), or cryopreserved (65.0 +/- 2.6%). Freshly isolated hepatocytes secreted more albumin after transplantation than hepatocytes that were cultured, hypothermically stored, or cryopreserved. In conclusion, culture and storage profoundly compromises the function of isolated hepatocytes after transplantation. Freshly isolated hepatocytes are the preferred source for transplantation.