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

正在开发新的方法来创建能够模拟3D自然人体组织环境的体外模型，以研究受控系统中的细胞生物学行为。动物模型、细胞培养和患者活检增加了我们对人类唾液腺(HuSG)生物学的理解。然而，它们只提供了关于huSG如何发挥作用、修复和再生的有限证据。建立像人工huSG系统这样的体外模型可以实时提高我们对huSG生物学的理解。设计人工HuSG的挑战是1)在保持其分化状态的同时扩大唾液分泌细胞，2)HuSG组织短缺，3)为它们提供准确的支撑支架。因此，尽管我们在设计人造HuSG方面取得了进展，但我们仍然缺乏对如何将唾液细胞组装成功能齐全的组织的了解。我们5年研究的主要目标是设计一个健康的生理唾液腺系统，以实时研究HuSG生物学。为了实现这一目标，目标1将开发一种3D基质(生物墨水)，通过添加不同的细胞外基质(ECM)成分来模仿huSG天然组织。我们有了一个强大的ECM模拟矩阵的初步结果，该矩阵允许SG细胞重组为球体(体内组装器官的原始单元)。由于每次实验都没有足够的新鲜组织(活组织检查)，Aim#2将使huSG细胞系永生化，以提供稳定和可重复的huSG细胞来源。我们有一个永生的细胞系，在我们的3D基质中作为健康的、有活力的球体生长。目标3号将设计一种人工3D系统，在体外模拟huSG细胞组织。使用计算机辅助设计模型、生物墨水和生物打印技术，我们将在3D模型中的特定位置打印不同类型的细胞。在初步研究中，我们已经打印了一个原型，使用两种不同类型的细胞和唾液分泌细胞，尽管由于其重组为球体而尚未发挥功能。目标4将确定与唾液产生有关的关键蛋白质，以及它们在3D打印系统中的正确位置。我们将使用下一代技术和药物刺激来验证人造器官的功能(唾液产生)。最后，将功能性huSG构建作为一个有机扩展系统并移植到活体模型中，实现一个实时(长期目标)可信的SG生物学研究系统。这项研究计划将为huSG提供一个可靠、可重复的体外扩增系统，为实时了解SG生物学的高级研究创造一个新的平台，这也将有利于生理学、干细胞、细胞/组织工程和生物材料的研究。我们对计划中的计划抱有很高的期望，希望为细胞移植和再生医学带来无与伦比的见解。

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.