The effects of storage conditions and trophic supplementation on the survival of fetal mesencephalic cells

Abstract

It is estimated that only 5–10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K + hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37°C in HM killed all cells, while storage at 10°C yielded higher survival rates than 4°C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 × 10 6 cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 μg/mL), and brain-derived neurotrophic factor (BDNF; 10 μg/mL). GDNF and HPCS supplements increased RMT cell viability by 10–15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.