Isolation of mouse‐to‐rat cardiac xenograft‐infiltrating cells by ex vivo propagation

Ex vivo propagation of graft infiltrating lymphocytes has become a useful method for the examination of the cellular response after allogeneic transplantation. The aim of the present study was to investigate if this method can be used also for isolation of xenograft infiltrating cells, and, if so, to further characterize these cells. The concordant mouse-to-rat heart transplantation model was used for the experiments. Recipient rats were treated either with 15-deoxyspergualin (DSG) or with a combination of DSG and cyclosporine A (CyA) or left untreated. Transplants were removed beating on day 2 (untreated) or day 8 (DSG and CyA + DSG) and biopsies were incubated in culture medium for 48 h, resulting in propagation of cells from the biopsies into culture medium. The propagated cells were counted and phenotyped using flow cytometry. In parallel, the transplants were examined morphologically and immunohistochemically. Infiltrating cells could be isolated from all grafts in all groups. The number of propagated T lymphocytes during cellular rejection (DSG-treatment) was about 3.5 times higher than during 'non-rejection' (CyA + DSG) and six times higher than during acute vascular rejection (untreated). These findings were supported both by the morphological and the immunohistochemical findings. In conclusion, we have shown that graft infiltrating lymphocytes can be isolated from xenogeneic heart transplants by incubation of biopsies for 48 h, resulting in spontaneous propagation of immune cells into culture medium. Propagated cells could be further characterized by flow cytometry. Thus, the technique presented here can be used as a tool for studies of xenogeneic cellular rejection.