Intracerebral transplantation of cultured neurons after reaggregation in a plasma clot.
In order to be able to transplant neural cells which have been either manipulated in vitro or maintained in culture for the purpose of cell-type enrichment, we have developed a novel plasma clot method which permits reaggregation of previously dissociated cells such that they can be implanted as highly localized transplants rather than as dispersion-prone cell suspension grafts. To establish the method, enzymatically dissociated cells prepared from hippocampal primordia of late embryonic rats were immediately reaggregated into plasma clots and transplanted to the hippocampal formation of adult recipients. By using fluorescein-labelled bovine plasma to form the plasma clot grafts of reaggregated cells, the fate of the plasma clot protein matrix was followed at different post-operative survival times. Initially, 4-5 days post-operative, the plasma clot maintained the grafted cells in a loose sponge-like sack at the site of implantation. After 2-3 weeks, the transplanted cells were more compact and fused with the host neuropil, and the plasma clot matrix had largely been degraded. At 1 month or longer survival, there was no distinguishable boundary between transplant and host, and there was little or no evidence of any remaining plasma clot matrix or proteins. The plasma clot method was subsequently applied to the transplantation of cultures enriched in pyramidal cells. Enrichment for pyramidal cells was achieved by eliminating mitotic cells (dentate granule cells and glia) by brief (200 rad) irradiation of hippocampal primordia followed by dissociation and maintenance in monolayer culture for 4-6 days. Fibres from host dentate granule cells grew into the pyramidal cell-enriched transplants and established mossy fibre terminals on the donor cells. In transplants between embryonic and adult rats, donor cells were identified at long survival times by prelabelling donor cells in utero or in vitro with [3H]thymidine prior to transplantation. In transplants between embryonic and adult mice, donor tissue from A Thy-1.1 strain mice was transplanted to congenic A strain (Thy-1.2) mice such that the donor cells bearing the Thy-1.1 cell surface glycoprotein could be later identified by immunocytochemical staining with antibodies specific for the Thy-1.1 antigen. Reaggregation and transplantation of dissociated cells in a plasma clot thus provides a novel method whereby prior manipulation of neural tissue (separation of neurons and glia, enrichment for specific types of neurons, or glia etc.) can be used to great advantage in studying host-transplant connectivity and in assessing those factors which are critical in sustaining the survival of grafted neural tissue.
D000818:Animals; D001777:Blood Coagulation; D002449:Cell Aggregation; D002478:Cells, Cultured; D006085:Graft Survival; D006624:Hippocampus; D008722:Methods; D009474:Neurons / Q000637:transplantation*; D010949:Plasma; D051381:Rats