A Comparative Study of Preparation Techniques for Improving the Viability of Nigral Grafts using Vital Stains, in Vitro Cultures, and in Vivo Grafts

Abstract

The intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells. Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation. We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies. For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts. The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported. In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension. The optimal preparation technique, therefore, uses E13-E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette. This may have important implications in improving intracerebral transplantation for Parkinson's disease.