Rodysill et al. (2012). Liver Transplantation. doi: 10.1002/lt.23598
…minutes. RPMI-1640 (10 lL) was added to each well, and they were incubated at room temperature for 10 minutes. The supernatant from each well was removed. Two-microliter aliquots of a rabbit comple- ment (Pel-Freez, Rogers, AR) diluted 1:10 in RPMI- 1640 were added to each well, and they were incu- bated at 37C for 30 minutes. A fluorescence-acti- vated cell sorting buffer containing formalin (20 lL) was added to…
Pel-Freez products cited: Baby Rabbit Complement (31062)
Abstract
In utero cell transplantation (IUCT) can lead to the postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCT have involved hematopoietic stem cells. It is unknown whether human hepatocytes used for IUCT in fetal pigs will lead to the engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1 × 10 7 human hepatocytes in utero and were delivered by cesarean section at term. The piglets received a second direct liver injection of 5 × 10 7 human hepatocytes 1 week after birth. The serum was analyzed for human albumin 2, 4, and 6 weeks after engraftment. Piglet livers were harvested 6 weeks after transplantation and were examined by immunohistochemistry, polymerase chain reaction, and fluorescence in situ hybridization for human-specific sequences. Piglets undergoing IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all postengraftment time points. Human albumin gene expression, the presence of human hepatocytes, and the presence of human beta-2 microglobulin were all confirmed 6 weeks after engraftment. IUCT in fetal pigs with human hepatocytes early in gestation allowed the engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large-scale expansion of human hepatocytes in genetically engineered pigs.