Long-term survival of dopaminergic neurones in free-floating roller tube cultures of human fetal ventral mesencephalon

Abstract

Transplantation of human fetal ventral mesencephalon (VM) to Parkinsonian patients has shown beneficial effects in several clinical trials. However, further improvements in the transplantation technique are needed. Delayed surgery, i.e., the in vitro maintenance of the tissue prior to transplantation would present several advantages. The roller tube technique as initially described by Gähwiler (1981) was modified in several aspects for the long-term maintenance of dopaminergic neurones of human fetal VM. Tissue cultures were maintained free-floating in the medium for up to 42 days. The human fetal material was obtained from legal induced suction abortions. The embryonic age ranged from 5 to 12 weeks post-conception. Identification of VM was possible in 43% of the cases. Neurones in cultures were demonstrated by means of immunohistochemistry for tyrosine hydroxylase (TH) and γ-amino butyric acid (GABA), by electron microscopy and by hybridisation histochemistry using a TH-mRNA-sensitive probe. A high variability in the number of TH-positive cells in individual cultures derived from the same embryo was observed. In 20 μm frozen sections of such tissue cultures the mean ± SEM of TH-positive cells was 6.5 ± 1.2 0.1 μ m 2 ( n = 79; range: 0–73). The technique described insures the growth of long-term cultures of human fetal VM.