PRESERVATION OF FETAL VENTRAL MESENCEPHALIC CELLS BY COOL STORAGE - IN-VITRO VIABILITY AND TH-POSITIVE NEURON SURVIVAL AFTER MICROTRANSPLANTATION TO THE STRIATUM

Preservation of fetal ventral mesencephalic (VM) dopaminergic tissue p rior to transplantation has been hampered by the fact that the cells a re vulnerable to mechanical and osmotic stress after storage. Previous quantitative studies have shown that cool storage in a so-called 'hib ernation medium' prior to grafting, can be used safely for up to 2 day s without morphological or functional losses [16,32] using standard tr ansplantation techniques. In the present study on rat fetal VM tissue we have investigated (i) the accuracy of different vital stains (trypa n blue exclusion and ethidium bromide stain) to predict in vivo viabil ity of VM cell suspensions after grafting; (ii) the influence of diffe rent storage media (glucose-saline, HBSS, DMEM, CO2-independent medium and hibernation medium), temperatures (+4 degrees C or +21 degrees 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 preserva tion by cool storage for up to 12 days using a less traumatic microtra nsplantation technique. The results show that cool storage at +4 degre es C of intact VM pieces in hibernation medium gives the best in vitro viability scores. Microtransplantation of cell suspensions prepared f rom cool-stored VM tissue produced good survival of tyrosine hydroxyla se (TH)-positive graft neurons for up to 8 days of storage, and functi onal compensation in the amphetamine-rotation test for up to 12 days o f storage. The total yield of surviving TH-positive neurons was unchan ged, 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 o f cool storage and microtransplantation techniques to be able to exten d the preservation periods of VM tissue. Such procedures may ultimatel y help to increase the safety and flexibility in experimental and clin ical studies on neural transplantation of dopaminergic neurons.