Culture of Enzymatically Dispersed Anterior Pituitary Cells: Functional Validation of a Method

Abstract

A method for preparing short-term enzymatically dispersed pituitary cell cultures is described. It has been used to assay hypothalamic releasing factors. The cells secrete TSH or LH and FSH in amounts directly related to the dose of TRF or LRF respectively. The minimal active doses of synthetic TRF and either synthetic or highly purified ovine LRF is ca. 10~1 0 M. Apparent affinity constants for TRF or LRF receptors are ca. 10~°M. Enhanced secretion rates of TSH or LH of 8-to 20-fold in response to maximal levels of TRF or LRF are routinely observed. The sensitivity of the cultured cells to TRF and LRF is maintained for more than 2 weeks in culture although the magnitude of the secretory response and the intracellular hormone content declines with time in culture. Chronic administration of TRF or LRF depletes the intracellular hormone levels while simultaneously increasing the total TSH or LH content in the culture (tissue and fluid). As has been observed with other in vitro systems, high medium potassium, prostaglandin E 2 and theophylline stimulate TSH secretion from the cultured cells. Furthermore, thyroid hormones which suppress the TSH secretion in response to TRF by the anterior pituitary in vivo and in other in vitro methods are similarly effective on these cells. The use of this technique has been applied to pituitary tissues of other species and types of cells, e.g., cells from Gj mouse thyrotropic tumors were shown to respond appropriately to TRF and thyroxine. The enzymatically dispersed cultured cells have been found to behave as would have been expected on the basis of experience with other in vitro methods while having significant advantages over those other procedures.

Precursor—product relationships in the biosynthesis and secretion of thyrotropin and its subunits by mouse thyrotropic tumor cells

“…Cells were enzymatically dispersed by the method of Vale et al [8] and suspended in Dulbecco's Modified Eagle's Medium supplemented with 10% hypothyroid calf serum (Rockland Farms), 10 mM Hepes, 2 mM glutamine, 100 U/ml penicillin, and 100 /.&nl streptomycin. One ml of cell suspension (approx.…”

The Interactive Effects of VIP, PHI, GHRH and SRIF on the Release of Growth Hormone from Cultured Adenohypophysial Cells in Cattle.

“…Adenohypophysial cells were enzymatically dispersed by a method previously described by Vale et al [16] and modified in our laboratory [19,20]. The cells (5 x 104 cells/ml) resuspended in Dulbecco's modified Eagle's medium (DMEM) [20] were plated in Costar 24-well dishes and maintained at 37 °C in a humidified atmosphere of 5% CO2.…”

The Effect of Interleukin-2 on the Release of Gonadotropin and Prolactin in Vivo and in Vitro.

“…The posterior lobes of pituitary glands were removed and the anterior lobes were dispersed by a modified method of Vale et al [20]. The dispersed cells were aliquoted (2.5 X 105 cells/ml) into 24-well tissue culture plates and preincubated at 37°C under 5% CO2-95% air in Earle's medium 199 (M 199, Gibco) containing 25 mM Hepes, 10% horse serum, penicillin and streptomycin.…”