Comparison of the Construction Methods of Parathyroid Organoids and Functional Influence of Calcium on Organoids

ABSTRACT Objective This study aimed to determine the more suitable method for constructing parathyroid organoids. Methods Parathyroid organoids were established using two different culture conditions, and organoid morphology and ATP activity were assessed to evaluate their performance. Organoid identity and function were confirmed by STR profiling, histology, immunofluorescence, and ELISA. Organoids were further treated with different calcium concentrations, and changes in morphology, ATP activity, intracellular calcium, CaSR expression, and PTH secretion were examined. Results Organoids cultured in VEGF‐121‐ and angiotensin‐negative medium showed no noticeable morphological change from Day 1 to Day 3, and ATP activity declined over the same period. In contrast, organoids in VEGF‐121+ and angiotensin‐positive medium exhibited increased number and ATP activity over the same period. These organoids shared the same mutation profile and structural features as the original tissues and expressed PTH, GCM2, CaSR, and CHGA. PTH secretion remained stable across passages. Low calcium had minimal impact on morphology, viability, and PTH release but reduced intracellular calcium levels and CaSR expression. High calcium promoted ATP activity and elevated intracellular calcium, while suppressing PTH secretion and upregulating CaSR expression. Conclusions VEGF‐121+ and angiotensin‐positive medium better supported the generation and maintenance of functional parathyroid organoids. This provides an in vitro model for further investigation into parathyroid‐related signaling and applications in regenerative medicine.