Preservation of fetal ventral mesencephalic cells by cool storage: in-vitro viability and TH-positive neuron survival after microtransplantation to the striatum

Abstract

Preservation of fetal ventral mesencephalic (VM) dopaminergic tissue prior to transplantation has been hampered by the fact that the cells are vulnerable to mechanical and osmotic stress after storage. Previous quantitative studies have shown that cool storage in a so-called ‘hibernation medium’ prior to grafting, can be used safely for up to 2 days without morphological or functional losses [16,32] using standard transplantation techniques. In the present study on rat fetal VM tissue we have investigated (i) the accuracy of different vital stains (trypan blue exclusion and ethidium bromide stain) to predict in vivo viability of VM cell suspensions after grafting; (ii) the influence of different storage media (glucose-saline, HBSS, DMEM, CO 2-independent medium and hibernation medium), temperatures (+4° C or +21°C) and preparations (cell suspension or intact pieces) on the viability scores and total number of cells in vitro; and (iii) the survival and functional effects of intrastriatally grafted VM tissue after preservation by cool storage for up to 12 days using a less traumatic microtransplantation technique. The results show that cool storage at +4°C of intact VM pieces in hibernation medium gaves the best in vitro viability scores. Microtransplantation of cell suspensions prepared from cool-stored VM tissue produced good survival of tyrosine hydroxylase (TH)-positive graft neurons for up to 8 days of storage, and functional compensation in the amphetamine-rotation test for up to 12 days of storage. The total yield of surviving TH-positive neurons was unchanged, compared to fresh grafts, after 5 and 8 days of storage, and only reduced by 48% in the grafts stored for 12 days prior to implantation. These findings highlight the potential usefulness of a combination of cool storage and microtransplantation techniques to be able to extend the preservation periods of VM tissue. Such procedures may ultimately help to increase the safety and flexibility in experimental and clinical studies on neural transplantation of dopaminergic neurons.