Preparation and culture of dispersed avian pituitary cells, and age-related changes in donor pituitary weight and growth hormone content

Abstract

Techniques were perfected for the enzymatic dissociation of chicken pituitary glands and a number of factors evaluated for their effects upon growth hormone (GH) production by dispersed chicken pituitary cells in culture. Age-related changes in donor pituitary weight and GH content were also determined. A procedure involving digestion of minced glands with a solution of 0.1% trypsin in S-MEM tissue culture medium (0.1% BSA) for 1 hr at 37° under an atmosphere of 5% CO 2-95% air yielded >2.0 × 10 6 cells per gland with 80–90% viability. Five tissue culture media (D-MEM, α-MEM, RPMI 1640, Med-199, Earle's salts), two serum sources (calf serum (CS), horse serum (HS), and two levels of serum (5, 20%) were tested for their ability to support GH synthesis over 4 days in culture. Additionally, two culture regimes (continuous culture vs daily media changes) were evaluated for their effects on GH production. α-MEM resulted in the numerically highest net GH synthesis (over starting cell content), although not statistically different from RPMI 1640 or Earle's salts. Neither serum type nor percentage was significant; therefore the lower serum percentage (5) was adopted for future studies. Culture regime significantly altered the proportion of secreted vs stored hormone harvested at the end of the culture period. Changing media daily resulted in a 40% reduction in final cell GH content compared to continuous culture, whereas total cumulative media GH was approximately 39% greater ( P < 0.01). Pituitary weight increased with age until approximately 9 weeks, whereas GH content plateaued carlier, at 5 weeks of age. Thus, maximum pituitary GH concentration occurred at 5 weeks of age (28 μg/mg pituitary wet wt) in birds studied here. These results may have implications for future studies of avian GH synthesis and release utilizing dispersed avian pituitary cells in culture, as well as considerations for in vivo work.