Characteristics of bovine parathyroid cell organoids in culture

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Abstract
Adult bovine parathyroid glands were enzymatically dispersed and groups of 2 to 5 million cells were reassociated into multicellular aggregates (organoids) by rotation in roller tubes in serum-free medium. Fifty to seventy percent of the seeded cells were incorporated into each organoid at 3 d of culture, and in a typical experiment where DNA content was assayed before and after culture 49 ± 3% of the original seeded DNA was present after 19 d of culture. No significant differences in DNA content were observed between experimental groups at any time of culture. The morphology of the cells in organoids was similar to that of cells in fresh tissue as determined by light and electron microscopy. The organoids secreted intact parathyroid hormone (PTH) and COOH-terminal hormone fragments which were similar to those released from monolayer cell cultures. Organoids maintained the ability to modulate PTH secretion in response to extracellular calcium for over 2 wk in culture. Each organoid was cultured separately and secreted PTH such that the mean standard deviation of secretion within groups on a per organoid basis was 16.3% of the mean. Using a perifusion system to study acute regulation over a 2-wk period of culture, PTH secretion was suppressed 58±4% by 2.5 mM compared to that at 0.25 mM calcium. To examine PTH secretion over a range of calcium concentrations, the perifusion system was used to apply 4-h linear gradients of decreasing calcium to fresh tissue slices and to organoids. The results indicated that the calcium (ionized) concentration at 50% secretory suppression (set-point) were 1.30±0.11 and 1.20±0.9 mM for the organoids and slices, respectively. Acute secretory control by calcium decreased after 14 d and was not detectable at 22 d of culture. The results demonstrated that the organoids maintained their differentiated function and tissuelike morphology for extended periods in vitro and therefore represent a suitable model system for studies on the long-term modulation of PTH secretion by vitamin D metabolites, ions, and other agents.