Student Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China., China (People's Republic)
Abstract: The development of organoid research provides new possibilities for regenerative medicine and translational applications. Organ and tissue reconstruction based on organoid research, as well as drug screening chips leveraging organoid technology, hold significant clinical and scientific value. However, achieving precise regulation of organoid proliferation and differentiation remains a critical bottleneck for large-scale clinical translation. Here, we present a novel microwell array-based three-dimensional culture system, which enables uniform production of salivary gland organoids in size and differentiation status through microenvironment regulation. In this system, AggreWell™800 was used to create a PDMS microwell array to mold an agarose culture dish. The agarose surface was coated with Matrigel, resulting in a 3D culture system featuring uniform trapezoidal microwells. Salivary gland organoids were self-assembled by co-culturing human minor salivary gland stem cells (hMSG-SCs) and human minor salivary gland mesenchymal stem cells (hMSG-MSCs). In the microwell array culture system, organoids with a uniform diameter of 223.74 ± 18.36 μm were obtained on day 3. Molecular biology analyses, including qPCR and Western blot, revealed that compared to organoids generated in traditional Matrigel-based 3D culture system, organoids derived from this system exhibited enhanced expression of stem cell markers (LGR5, SOX2) and proliferation marker KI67, accompanied by a significant reduction in differentiation-related acinar markers (AMY1B, AQP5) and ductal markers (KRT7, KRT19), suggesting better preservation of stem cell characteristics. This innovative approach offers a promising strategy for generating "seed organoids" with enhanced stemness and controllable differentiation capacity. This culture system enables large-scale production of early-stage organoids and allows precise regulation of proliferation and differentiation by adjusting microwell size and biological culture conditions, supporting personalized configurations for organ reconstruction or chip fabrication.
Funding Source: This work was funded by the National Nature Science Youth Foundation of China (Grant Number 81801946).
