Endocrinology, Vol 115, 1406-1411, Copyright © 1984 by Endocrine Society

ARTICLES

Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation

H Chamras, JM Hershman and TM Stanley

To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5- fold; there was no significant increase in TSH release in fractions 3- 6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.