Bovine parathyroid cells: cultures maintained for more than 140 population doublings.

Abstract

Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release.Short-term studies with dispersed cells or tissue explants in vitro have yielded considerable information about mechanisms controlling secretion ofparathyroid hormone (PTH) (1) but, unlike well-defined cell culture systems, are not suitable for studies of control of parathyroid cell proliferation and differentiation. Hormone-producing parathyroid cells have been maintained as mixed cell types in culture for short periods but have replicated poorly and eventually become overgrown by fibroblasts (2, 3). The major impediment to effective study of parathyroid cell growth has been the lack of methods for long-term growth of these cells. We describe here a system for primary culture and sustained growth (> 140 population doublings) of differentiated bovine parathyroid cells using serum-coated Petri dishes for cell attachment and a complex serum-free medium for growth. Totally serum-free culture is possible using collagen or fibronectin as a coating for the plates. MATERIALS AND METHODSPrimary Cultures. Bovine parathyroid glands were collected on ice immediately after slaughter and brought to the laboratory in buffer A (20 mM Hepes, pH 7.45, in NIH Eagle's medium 2 without bicarbonate)/1 mM Ca2+/1 mM Mg2+ containing 500 units of penicillin, 500 ,ug of streptomycin, and 100 jig of gentamycin per ml. The glands were sterilized by stirring for 1 min in 0.5% Pheno-cen (Central Chemical, Kansas City, KS) in saline. The tissue was trimmed, minced into fragments, and washed twice in 8 mM sodium phosphate/138 mM NaCl, pH 7.2 (phosphate-buffered saline). One gram of tissue was suspended in 10 ml of buffer A containing 500 ,ug of DNase I (Sigma), 15 mg of collagenase type II (Worthington), 10 mg of glucose, 30 mg of bovine serum albumin type IV, 0.3 mM CaCl2, 0.5 mM MgCl2, and 2% heat-inactivated dialyzed chicken serum. Tissue fragments were digested at 37°C in humidified 95% air/5% CO2 for 3 hr, pipetting every 30 min with a plastic serological pipet. The mixture, washed three times with phosphate-buffered saline by alternately centrifuging at 500 x g and dispersing mechanically, was filtered through 60-pim and 15-,um Nitex mesh screens (Tetko, Elmsford, NY). The small groups of cells that passed through the 60-,um but were retained on the 15-,um filter were collected, suspended in complete GSmF12 medium (see below), and distributed into bov...

Mechanisms of secondary hyperparathyroidism

“…After 24 h in culture, CaR mRNA and protein are present only in very low concentrations on the PT cells in primary culture, and at later time intervals, not present at all. There have been studies in a cell line derived from rat PT showing that Ca 2ϩ regulates PT cell proliferation (11) and there are changes in cyclin D1 mRNA, not cyclin D2 and D3, after changes in medium Ca 2ϩ concentration (7). Sakaguchi et al (62,63) showed that these cells expressed acidic fibroblast growth factor (aFGF) and expression of both aFGF mRNA and peptide was suppressed by calcium.…”

Quantitative and three‐dimensional aspects of the rat parathyroid gland in normo‐, hypo‐, and hypercalcemia

“…Upon hypocalcemia, synthesis is markedly increased within 6 hours. This can hardly be explained by cell hyperplasia, since the cell cycle measured in vitro is approximately 20 hours (Brandi et al, 1986). In studies of long-term (3 weeks) hypocalcemia, a fivefold increase in PTH mRNA levels was observed despite high endogenous calcitriol levels, indicating a dominant effect of hypocalcemia on PTH synthesis (Naveh-Many and Silver, 1990).…”

Serum‐free mouse embryo cells: Growth responses in vitro

“…Replacing serum in the culture medium by growth factors and other supplements allows extended culture of some cell types that cannot be maintained on a long-term basis in conventional serumcontaining media (Ambesi-Impiombato et al, 1980;Orly et al, 1980;Gospodarowicz et al, 1981;Hammond et al, 1984;Brandi et al, 1986). We have applied a similar serum-free approach to the culture of mouse embryo cells.…”