Organotypic Spheroid Culture to Mimic Radiation-Induced Salivary Hypofunction

Radiation treatment often leads to irreversible damage to normal salivary glands (SGs) because of their proximity to head and neck cancers. Optimization of the in vitro model of irradiation (IR)-induced SG damage is warranted to investigate pathophysiology and monitor treatment outcome. Here, we present an organotypic spheroid culture model to investigate the impact of IR on SGs and the mechanisms underlying IR-induced structural and functional changes. Human parotid epithelial cells were obtained from human parotid glands and plated on either plastic plates or Matrigel. A number of 3-dimensional (3D) spheroids were assembled on Matrigel. After IR at 10 and 20 Gy, morphologic changes in cells in 2D monolayers and 3D spheroids were observed. As the structural integrity of the 3D spheroids was destroyed by IR, the expression levels of salivary epithelial and structural proteins and genes decreased proportionally with radiation dosage. Furthermore, the spheroid culture allowed better measurement of functional alterations following IR relative to the monolayer culture, in which IR-inflicted spheroids exhibited a loss of acinar-specific cellular functions that enable Ca²⁺ influx or secretion of α-amylase in response to cholinergic or β-adrenergic agonists. p53-mediated apoptotic cell death was observed under both culture conditions, and its downstream signals increased, such as p53 upregulated modulator of apoptosis (PUMA), Bax, cytochrome c, caspase 9, and caspase 3. These results suggest that the organotypic spheroid culture could provide a useful alternative model for exploration of radiobiology and mode of action of new therapies for prevention of radiation-induced salivary hypofunction.