明胶微球因其高比表面积、良好的生物相容性和可控降解的特性在生物医学领域得到了广泛的应用，而尺寸均匀程度在这些应用中起着关键作用。但是传统的明胶微球制备方法不能满足尺寸均匀、大小可控的要求（直径变异系数大于20%），新兴的制备方法设备成本极其高昂，阻碍了其推广和应用。为解决该难题，本研究搭建了两种低成本的制造系统，一种基于电喷，一种基于共流型微流控，以实现均一化明胶微球的制造，并评估了上述工艺的可行性和可靠性。通过系统的优化，两套设备均可制造尺寸均一的明胶微球（直径变异系数小于5%）。但是简略的电喷设备无法精准控制某些参数（例如电场强度），从而导致了较差的复现性；而微流控工艺能稳定制造明胶微球，且尺寸可调（直径范围250~450微米），因此后续实验采用了稳定性更高的微流控。共流型微流控系统具有成本低廉、稳定性高、可拆卸、可拓展的优势，以促进微球在实验室环境下的研究和应用。本文基于上述开发的明胶微球制造系统进行了多方面应用的探索：（1）干细胞扩增。间充质干细胞的相关研究和应用与日俱增，需要极大数量的细胞，然而平面培养效率低、操作繁琐、难以规模化。对此，本论文将明胶微球用于间充质干细胞的3D培养，利用微球高比表面积的优势，在较小体积内收获大量的细胞。明胶微球良好的生物相容性为细胞高效扩增提供了保障，本研究实现了5天内最高可扩增约15倍。尺寸均匀的微球为批次间稳定性提供了保障。并且基于微球的培养方案能在传代和收获等操作中大幅度减少试剂消耗和工作量，实现大规模、高效率的细胞扩增。本研究对经过微球扩增的间充质干细胞进行了生物学评估，表明干细胞维持了正常的形态、表面标志物的表达和多向分化潜能。（2）基于微球的组织工程。本研究对明胶微球上的间充质干细胞进行了诱导分化，结果表明干细胞在微球上依然可以分化成为成骨细胞、成脂细胞和成软骨细胞并行使相应的功能，该微组织具有组织修复的潜能。此外，本研究将载有干细胞的明胶微球融入生物墨水，经过生物打印成为更大体积的组织工程支架，并观察到细胞存活良好，证实了载细胞微球的可打印性。结合干细胞的分化潜能，该支架具有促进组织再生修复的前景。 综上所述，本研究通过搭建共流型微流控平台实现了尺寸均匀可控的明胶微球的制造，该技术具有稳定性高、成本低廉、可拓展的优势，所制备的明胶微球可用于干细胞的3D培养扩增和基于微球的组织工程。

Gelatin microspheres (GMs) have garnered considerable attention in the biomedical field due to their high surface-to-volume ratio, excellent biocompatibility and tunable degradation properties, while the size homogeneity of GMs is critical for their successful application. However, conventional fabrication methods for GMs have proven inadequate in achieving precise size control, with diameter variation coefficients exceeding 20%. Emerging techniques, while promising, necessitate high-cost equipment, impeding their widespread utility. To address this challenge, we developed two cost-effective systems for fabrication of GMs based on either electro-jetting or co-flow microfluidics, to improve uniformity and size tunability. These methods were also evaluated for their feasibility and reliability. Through parameter optimization, both sets of system achieved production of GMs with uniform size (diameter variation coefficient less than 5%). However, the rudimentary electro-jetting device exhibited poor reproducibility due to several uncontrollable parameters such as electric field strength. In contrast, the co-flow microfluidic platform demonstrated remarkable stability and provided precise control over size of GMs (ranging from 250 μm to 450 μm), which was utilized in subsequent experiments. Owing to its low cost, robustness, detachability, and expansibility, co-flow microfluidics is well-suited for laboratory-based research and applications of microspheres. Based on the GMs fabrication device (co-flow microfluidics), this thesis also explored several applications of GMs: 1) stem cell expansion. The demand for mesenchymal stem cells (MSCs) in both academic research and clinical applications is rapidly increasing, necessitating large-scale cell amplification. However, plane cultivation is inefficient, cumbersome and difficult to scale. Thus, this study employed GMs as microcarriers into 3D culture and expansion of MSCs, which enabled the harvesting of a large number of cells in a small volume due to their high specific surface area. Due to the excellent biocompatibility of GMs, we achieved an approximately 15-fold increase within five days. Microspheres with uniform size ensure consistency between batches. Additionally, the reduced reagent consumption and workload during passage and harvest operations facilitated efficient and large-scale cellular expansion. We evaluated the biological function of MSCs harvested from GMs and demonstrated that the cells maintained their normal morphology, expression of surface markers, and multipotency differentiation. 2) microsphere-incorporating tissue engineering. This research performed induced-differentiation on MSCs cultured on GMs and found that the cells retained the multipotency towards osteoblasts, lipoblasts and chondroblasts with corresponding biological functions, enabling tissue repair. Furthermore, this thesis employed MSCs-laden GMs as bioinks for construction of tissue engineering scaffolds with larger volumes and observed high cell viability. As previous demonstrated, MSCs on the GMs maintained multipotency, indicating that the scaffolds possessed the potential of tissue regeneration.In conclusion, we established a co-flow microfluidics platform and successfully fabricated homogenous and size-controllable GMs, which demonstrated the stability, cost-effectiveness and expansibility of the platform. These GMs could be utilized in MSCs 3D culture and expansion, as well as in microsphere-incorporating tissue engineering.