An improved method for the expression of TRH in serum-supplemented primary cultures of fetal hypothalamic cells

Abstract

Primary cultures of dispersed cells from fetal nervous tissue are extensively used for studying multiple neuronal properties. Analyses of the developmental expression of thyrotropin releasing hormone (TRH; pglu-his-pro-NH 2) biosynthesis in primary cultures of fetal dissociated hypothalamic cells have shown that cellular TRH levels per dish increase with time in culture, after a lag period of a few days, but do not attain the values observed in vivo, hampering its use as a model system for the study of peptide biosynthesis and release. We have demonstrated that homologous conditioned medium (CM) enhances TRH expression in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. We report here experimental conditions that allow the expression, during the second or third week in vitro, of higher cellular TRH levels than previously described in primary cultures of dissociated hypothalamic cells from 17-day rat fetuses. The medium used was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), vitamins, glucose, glutamine and insulin (DMEM-S). Cellular levels of TRH/mg protein increased with cell density between 1 and 2.7×10 6 cells per 35-mm dish. Addition of 10 −5 M cytosine arabinoside (CAr) at the 4th day in vitro (DIV) improved TRH cell content per dish compared to addition at 5 DIV; 2.5–5×10 −5 M bromodeoxyuridine added at seeding reduced cell survival and did not enhance TRH levels, in comparison to CAr-treated cultures. Addition of ascorbic acid (0.5–1×10 −4 M) increased TRH levels per dish. Substitution of DMEM by DMEM-F12 (1:1) did not improve TRH levels. Cellular levels of TRH, in Neurobasal plus B27 (a serum-free medium), were similar to levels in serum-supplemented media. In the optimized conditions, a small number of pro-TRH mRNA expressing cells (2% of total cells) was detected by in situ hybridization; 40% coexpressed the pro-protein convertase PC1 mRNA. Conditioning the medium, controlling glial proliferation, and adding ascorbic acid improved the expression of TRH in primary culture of hypothalamic cells in DMEM-S.