SEPARATION OF RAT PITUITARY THYROTROPHIC CELLS

: A method for the enrichment of live thyrotrophic pituitary cells is described. Pituitary glands of young male rats were removed into Earle's solution and dispersed in a 0.1% trypsin solution containing 0.5% bovine serum albumin, pH 7.2. Nylon fibres (25 microgram) were used for the separation of the thyrotrophic cells, by stringing them across a plastic frame which fitted a plastic Petri dish containing the cell suspension. The fibres were washed with light petroleum (b.p. 60--80 degree C) and carbon tetrachloride, hydrolysed with 3 M-HCL for 30 min at room temperature and washed with distilled water and phosphate-buffered saline (pH 7.2). The fibres were treated with thyrotrophin releasing hormone (TRH) alone or in the presence of soluble carbodiimide solution. After incubation for 1 h at room temperature, the fibres were transferred to a new Earle's medium and cells were released from the fibres by plucking them with a needle. The separated thyrotrophic cells were identified by radioimmunoassay and by electron microscopy. Using the above-mentioned methods, enrichment of thyrotrophic cells was obtained. Thus, the amounts of TSH, prolactin, LH and GH released, during 2 h of incubation, by 1.5 x 10(6) unseparated cells were 6.8 +/- 0.65, 4.1 +/- 0.47, 4.8 +/- 0.52 and 5.2 +/- 0.68 microgram respectively, while the same number of purified thyrotrophic cells released 76.1 +/- 0.42, 1.2 +/- 0.3, 0.6 +/- 0.35 and 1.6 +/- 0.22 microgram of the same hormones (means +/- S.E.M.).