In Vitro Cellular & Developmental Biology - Animal

, Volume 30, Issue 9, pp 596–603

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

Authors

  • Kedar N. Prasad
    • Center for Vitamins and Cancer Research, School of MedicineUniversity of Colorado Health Sciences Center
  • Erika Carvalho
    • Center for Vitamins and Cancer Research, School of MedicineUniversity of Colorado Health Sciences Center
  • Susan Kentroti
    • Department of Pharmacology and Psychiatry, School of MedicineUniversity of Colorado Health Sciences Center
  • Judith Edwards-Prasad
    • Center for Vitamins and Cancer Research, School of MedicineUniversity of Colorado Health Sciences Center
  • Curt Freed
    • Department of Medicine, School of MedicineUniversity of Colorado Health Sciences Center
  • Antonia Vernadakis
    • Department of Pharmacology and Psychiatry, School of MedicineUniversity of Colorado Health Sciences Center
Cellular Models

DOI: 10.1007/BF02631258

Cite this article as:
Prasad, K.N., Carvalho, E., Kentroti, S. et al. In Vitro Cell Dev Biol - Animal (1994) 30: 596. doi:10.1007/BF02631258

Summary

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 400µg/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 pSV3neo, 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.

Key words

immortalizationdopamine-producing cellshomovanillic acidT-antigenstyrosine hydroxylase

Copyright information

© Society for In Vitro Biology 1994