Protocol for the generation and analysis of organoids from Dental pulp stem cells (DPSCs)

DPSCs are a valuable resource for creating three-dimensional (3D) in vitro models, such as organoids, due to their accessibility and differentiation potential. Organoids have revolutionized in vitro culture, thanks to their ability to more faithfully replicate the architecture and complexity of tissues compared to 2D cultures. This enables their use in a wide range of applications, including research on genetic diseases, innovative therapies, and tissue engineering. This project aims to establish an in vitro protocol for generating 3D models from DPSCs to study dental diseases and test new drugs for regenerative medicine and tissue reconstruction. To this end, the organoids underwent morphological, viability, and functional analyses, allowing us to evaluate the effectiveness of the protocol and the validity of the model. The protocol includes the culture of DPSCs in Matrigel® Matrix, an extracellular matrix that supports 3D-fabricated organoids formation and growth. These 3D structures are exposed to differentiation factors to generate dental pulp organoids. However, the presence of Matrigel® Matrix may interfere with subsequent molecular analyses. To overcome this limitation, an efficient method for removing the Matrigel® Matrix was developed to allow efficient extraction of nucleic acids and proteins. Immunohistochemical and immunofluorescence techniques were also optimized to visualize cellular structures and markers. Cell count and proliferation were assessed using a CCK-8 viability assay, as well as Trypan Blue staining. The feasibility and effectiveness of this protocol can provide a new tool for studying dental pulp biology, paving the way for future applications in the field of dental pulp regeneration research.