Establishment and Characterization of Immortalized Clonal Cell Lines from Fetal Rat Mesencephalic Tissue

• Published in: In vitro cellular & developmental biology. Animal 1994-09, Vol.30A (9), p.596-603
• Main Authors: Prasad, Kedar N., Erika Carvalho, Susan Kentroti, Edwards-Prasad, Judith, Curt Freed, Vernadakis, Antonia
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antigens, Polyomavirus Transforming - analysis; Antigens, Polyomavirus Transforming - genetics; Cell growth; Cell Line, Transformed; Cell lines; Cells; Cellular Models; Clone Cells; Cultured cells; Drug Resistance - genetics; Fluorescent Antibody Technique; Mesencephalon - cytology; Mesencephalon - embryology; Mesencephalon - metabolism; Mice; Mice, Nude; Neomycin; Neoplasm Transplantation; Neoplasms, Experimental - etiology; Neuroglia; Neurons; Plasmids; Rats; Rats, Sprague-Dawley; Somatic cells; Tissue grafting; Transfection; Tyrosine 3-Monooxygenase - analysis; Viral tumor antigens
• ISSN: 1071-2690; 1543-706X
• DOI: 10.1007/BF02631258

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$400 \mug/ml$geneticin). The survivors showed the presence of T-antigens and were non-tumorigenic. Two cell lines,$1RB_3$derived from cells transfected with$pSV_{3^{neo}}$, and$2RB_5$derived from cells transfected with$pSV_{5^{neo}}$, 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.