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In Vitro Cell Dev Biol Anim. 1994 Sep;30A(9):596-603.

Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.

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Center for Vitamins and Cancer Research, School of Medicine, University of Colorado Health Sciences Center, Denver 80262.


This investigation reports for the first time the establishment of immortalized clones of dopamine-producing nerve cells in culture. Freshly prepared single-cell suspensions from fetal (12-day-old) rat mesencephalic tissue were transfected with plasmid vectors, pSV3neo and pSV5neo, using an electroporation technique. Cells were plated in tissue culture dishes which were precoated with a special substrate and contained modified MCDB-153 growth medium with 10% heat inactivated fetal bovine serum. The immortalized cells were selected by placing the transfected cells in a selection medium (modified MCDB-153 containing 400 micrograms/ml geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines, 1RB3 derived from cells transfected with pSV3neo, and 2RB5 derived from cells transfected with pSV5neo revealed only 1 to 2% tyrosine hydroxylase (TH)-positive cells. Repeated single-cell cloning of these cell lines by a standard technique failed to increase the number of TH-positive cells in any clones. Using three cycles of growth, alternating between hormone-supplemented, serum-free medium and serum-containing medium produced a cell line (1RB3A) that was very rich in TH-positive cells. The recloning of 1RB3A yielded clones some of which contained over 95% TH-positive cells. These cells produced homovanillic acid, a metabolite of dopamine, and may be useful not only for neural transplant but also for basic neurobiological studies.

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