微流控多核凝胶微球培养具有完整小肠内壁组织结构的小肠类器官

In the fields of pharmacy, medicine and metabolomics, finding ways to construct an in vitro model of small intestinal inner wall with complete structure, which is closely related to the small intestinal functions about digestion, absorption and metabolic, is an urgent problem to be solved. Small intestinal organoids can form a functional epithelial tissue structure of the small intestine, which provides a way for solving the above problem. However, in the present studies, there is a lack of methods for culturing small intestine organoids with blood vessels and lymph nodes to simulate the complete inner wall structures of the small intestine. Based on the applicant's previous studies about the distribution characteristics of aqueous two phases (ATPS) and the production of ATPS emulsion particle stabilizer, and by combining the ATPS system with the water-in-water-in-oil multiple emulsion technology, this project propose a scheme for the generation of small intestinal organoids with complete tissue structures: Using multi-layer nested capillary apparatus for preparing alginate and Matrigel mixed multi-compartment ATPS emulsions and then gelation, multi-core hydrogel microspheres that encapsulated small intestinal crypt cells, Peyer's patch cells and microvascular endothelial cells are prepared firstly. Then, the growth environment of real intestinal stem cells is simulated by the microfluidic chip, and the important factors on the proliferation and differentiation of small intestinal stem cells, such as the effects of cell density, cell ratio, extracellular matrix physical and chemical properties, fluid stress are investigated. Smooth implementation of this project will provide theoretical guidances and material basis to building the in vitro model of small intestinal inner wall with complete tissue structures rapidly and conveniently in the relevant studies.

在药学、医学和代谢组学等研究领域，如何构建与小肠消化、吸收和代谢功能密切相关的具有完整结构的小肠内壁体外模型是一个亟待解决的关键问题。小肠类器官能够形成功能化小肠内壁上皮组织结构，为解决上述问题提供了一定思路。然而，当前缺少能够形成包含血管和淋巴模拟完整小肠内壁的小肠类器官培养方法。本项目在申请人前期对双水相分配特性和双水相乳液颗粒稳定剂的研究基础上，结合双水相与油包水包水多重乳液技术，利用多层嵌套毛细管装置生成含有海藻酸和基质胶的多隔室双水相乳液，凝胶化制备出分区稳定的包裹小肠隐窝细胞、派尔斑细胞和小肠微血管内皮细胞的多核水凝胶微球，以此为模板引入微血管和淋巴结构；于微流控芯片中模拟出体内干细胞生长环境，考察细胞密度、细胞比例、胞外基质理化性质和流体应力等重要因素协同作用对小肠干细胞增殖和分化的影响，探寻其规律性；为快速、便捷地建立具有完整小肠内壁结构的小肠类器官提供理论指导和物质基础。

如何构建与小肠消化、吸收和代谢功能密切相关的具有完整结构的小肠内壁体外模型是一个亟待解决的关键问题。本项目系统研究了利用微流控双水相液滴技术结合海藻酸凝胶，产生结构和功能可控的微球用于体外小肠模型的构建。提出并设计了一种微流控双水相液滴技术结合生物矿化来产生胶体囊的方法用于生物活性分子的可控包封和释放。结果显示，可通过改变生成微流控双水相模板液滴的大小、调节生物矿化中脲酶的浓度及矿化时间等条件来改变胶体囊的壳层密集程度实现被包裹的过氧化氢酶的可控释放。进一步，利用该微球包裹硝化菌进行了含铵氮废水处理的应用研究。结果表明，与传统海藻酸凝胶球载体相比，胶体囊能灵活调控尺寸大小，具有更大的反应比表面积，在高pH值及环境温度下能够更好地保留微生物的活性，并且能够较大的提高污水处理的循环使用次数。此外，利用一种具有生物活性的高分子蛋白质共聚物颗粒（mPEG-BSA）来稳定双水相乳液并以此做为模板通过滴注法制备一种高度多孔可控的多孔海藻酸钙微球。由于双水相的高度生物相容性，细胞能够直接被包封在该多孔微球的多孔结构中，使得其生长、增殖和功能表达情况都优于普通海藻酸钙微球。进一步地，在双重海藻酸凝胶微球中，通过分区包裹结肠癌细胞、人脐静脉内皮细胞和3T3细胞，构建了血管化肠粘膜体外模型。结果显示，紧密连接蛋白和β-连环蛋白在该模型中有较高地表达，证明其紧密连接良好；同时，结肠癌上皮功能标志蛋白碱性磷酸酶，P-糖蛋白，多药耐药蛋白2和血管化标志蛋白VEGF，在该模型中都有明显地表达。此外，还研制了基于海藻酸和GleMA的多孔凝胶微球，优化了其批量制备的微流控系统参数；成功构建了具有孔内孔外精确分区的包裹肝癌和HUVEC两种细胞的3D共培养系统。在多孔凝胶微球初步形成了有血管生成的肝癌类器官，显著提高了肝癌类器官对经典模型药物（阿霉素和顺铂）的剂量敏感性。本研究对再生医学相关生物材料结构设计与功能调控研究提供理论与实践指导。