Hydrogels as a 3D ex vivo platform for cell expansion, organoid formation, and engineering an artificial human salivary gland.

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New methodologies are being developed to create in vitro models that can mimic the 3D native human tissue environment to study cell biological behavior in a controlled system. Animal models, cell culture, and patient biopsies have increased our understanding of human salivary glands (huSG) biology. However, they only provide limited evidence on how huSG function, repair, and regenerate. Building an ex vivo model such as an artificial huSG system can improve our understanding of huSG biology in real-time. The challenges in engineering an artificial huSG are 1) to expand saliva-secreting cells while maintaining their differentiated state, 2) shortage of huSG tissue, and 3) supply them with an accurate supporting scaffold. Thus, despite our progress in engineering an artificial huSG, we still lack the understanding of how to assemble salivary cells into a fully functional tissue. The principal goal of our 5-year research is to engineer a healthy physiological salivary gland system to study huSG biology ex vivo in real-time. To achieve it, Aim#1 will develop a 3D matrix (bioink) that mimics huSG native tissue by adding different extracellular matrix (ECM) components. We have preliminary results of a robust ECM analog matrix that allows SG cells to reorganize as spheroids (a primitive unit of an assembled organ in vivo). Because there is insufficient fresh tissue (biopsies) for each experiment, Aim #2 will immortalize huSG cell lines to provide a stable and reproducible source of huSG cells. We have one immortalized cell line that grows as healthy, viable spheroids in our 3D matrix. Aim #3 will engineer an artificial 3D system simulating the huSG cell organization ex vivo. Using computer-assisted design models, bioinks, and bioprinting technology, we will print different cell types in specific locations in our 3D model. In preliminarily studies, we have printed one prototype using two different cell types with saliva-secreting cells, although non yet functional due its reorganization as spheroids. Aim#4 will identify the key proteins involved in saliva production and their correct location in the 3D-printed system. We will validate the artificial organ functionality (saliva production) using next-generation techniques and drug stimulation. Finally, the functional huSG construct will be used as an organoid expansion system and transplanted into in vivo models, achieving a trustable system for SG biology studies in real-time (long-term aim). This research program will provide a reliable, reproducible, ex vivo expansion system for huSG, generating a new platform for advanced studies in understanding SG biology in real-time that will also benefit research in physiology, stem cell, cell/ tissue engineering, and biomaterials. We have high expectations in the planned program to yield unparalleled insights for cell transplantation and regenerative medicine.