中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (28): 4483-4488.doi: 10.12307/2022.302

• 药物控释材料 drug delivery materials • 上一篇    下一篇

制备负载细胞可注射微球及体外评价

何家辰1，2，刘  畅1，2，陈迟迟1，2，施  勤1，2   

  1. 1苏州大学医学部，苏州大学附属第一医院，江苏省苏州市  215006；2苏州大学骨科研究所，江苏省苏州市  215006
  • 收稿日期:2021-02-24 接受日期:2021-03-31 出版日期:2022-10-08 发布日期:2022-03-21
  • 通讯作者: 施勤，研究员(正高)，苏州大学医学部，苏州大学附属第一医院，江苏省苏州市 215006；苏州大学骨科研究所，江苏省苏州市
  • 作者简介:何家辰，男，1996年生，安徽省六安市人，汉族，苏州大学在读硕士，主要从事免疫材料方面的研究。
  • 基金资助:
    国家自然科学基金面上项目(81772313)，项目名称：基于钛亲和性生物模拟活性肽双向调控骨形成和骨吸收促进钛植入材料骨整合效应的研究，项目负责人：施勤

Preparation and in vitro evaluation of injectable microspheres loaded with cells

He Jiachen1, 2, Liu Chang1, 2, Chen Chichi1, 2, Shi Qin1, 2   

  1. 1Medical College of Soochow University, First Affiliated Hospital of Suzhou University, Suzhou 215006, Jiangsu Province, China; 2Institute of Orthopedics, Suzhou University, Suzhou 215006, Jiangsu Province, China
  • Received:2021-02-24 Accepted:2021-03-31 Online:2022-10-08 Published:2022-03-21
  • Contact: Shi Qin, Researcher, Medical College of Soochow University, First Affiliated Hospital of Suzhou University, Suzhou 215006, Jiangsu Province, China; Institute of Orthopedics, Suzhou University, Suzhou 215006, Jiangsu Province, China
  • About author:He Jiachen, Master candidate, Medical College of Soochow University, First Affiliated Hospital of Suzhou University, Suzhou 215006, Jiangsu Province, China; Institute of Orthopedics, Suzhou University, Suzhou 215006, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 81772313 (to SQ)

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摘要：

文题释义：
甲基丙烯酰化明胶：是一种新颖的水凝胶材料，由甲基丙烯酰胺与明胶聚合形成的，明胶是明胶蛋白水解得到的重要衍生物，主要成分为胶原蛋白、蛋白多糖和部分氨基酸序列，其中精氨酸(Arg，R)-甘氨酸(Gly，G)-天冬氨酸(ASP，D)序列(即 RGD 序列)有利于细胞的黏附、增殖及分化。加入甲基丙烯酰胺后形成的甲基丙烯酰化明胶在明胶的基础上又显著提高了热稳定性。
微流控技术：具有精确的流体控制和高水平的平台整合等优点，利用多相液流的精确操控，微流控技术为纳米粒子、微纤维和三维(3D)结构的高通量合成提供了具有集成小型化与自动化特征的强大工具。该文开发的“Y”形通道用于制备液滴(圆形)的生物材料，结构均一，可大规模制备。

背景：利用生物材料作为细胞载体能够提供3D培养微环境，支持细胞活力和功能，并有可能扩大细胞治疗的数量和治疗效果，因此寻找一个合适的生物材料显得十分重要。
目的：制备可注射甲基丙烯酰化明胶多孔水凝胶微球，探究其生物相容性及负载细胞用于组织工程的潜力。
方法：利用微流控技术制备可注射甲基丙烯酰化明胶多孔水凝胶微球，表征微球的微观形貌与硬度。分别采用正常培养基(对照组)与微球浸提液(实验组)培养MC3T3-E1细胞，利用CCK-8法检测细胞增殖，活死细胞染色法检测细胞存活。将微球与CD3+T细胞共培养，以单独培养的CD3+T细胞为对照，光学显微镜下观察微球是否对T细胞活化产生影响，Dapi染色观察微球负载T细胞的形态，流式细胞术验证与微球共培养是否对CD4+T细胞和CD8+T细胞的比例产生影响。
结果与结论：①倒置显微镜下可见，冻干前后的微球均保持高度分散且大小均一，直径大小满足可注射条件；扫描电镜下可见，冻干后的微球为多孔结构，孔隙均匀分布；微球的弹性模量为(9.76±2.04) kPa。②CCK-8检测与活死细胞染色显示，两组MC3T3-E1细胞的增殖趋势与增殖活性无明显差别。③光学显微镜下可见，培养2 d时微球未引起CD3+T细胞的活化，也未干预CD3+T细胞的活化，CD3+T细胞分布于微球表面及孔隙内。④流式细胞术检测显示，微球未影响CD4+T细胞和CD8+T细胞的比例。⑤结果表明，通过微流控技术制备了一种可注射甲基丙烯酰化明胶多孔微球，其具有良好的生物相容性且不会对细胞产生不利影响，是一种在组织工程中具有广阔应用前景的生物材料。
缩略语：甲基丙烯酰化明胶：gelatin methacryloyl，GelMA

https://orcid.org/0000-0001-5835-2912(何家辰) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 微流控, 水凝胶, 多孔微球, 可注射, 生物相容性, 细胞疗法, T细胞, 组织工程

Abstract: BACKGROUND: The use of biological materials as cell carriers can provide a 3D culture microenvironment, support cell viability and function, and may expand the number and therapeutic effects of cell therapy. Therefore, it is very important to find a suitable biological material.
OBJECTIVE: To prepare injectable gelatin methacryloyl porous microspheres, and explore their biocompatibility and the potential of loaded cells for tissue engineering.
METHODS: Injectable gelatin methacryloyl porous microspheres were prepared by microfluidic technology. The microscopic morphology and hardness of microspheres were characterized. MC3T3-E1 cells were cultured in normal medium as control group and microsphere extract as experimental group. Cell proliferation was detected by CCK8 assay. Cell survival was detected by live dead cell staining. Microspheres were co-cultured with CD3+ T cells. CD3+ T cells cultured alone were used as controls. The effect of microspheres on T cell activation was observed under light microscope. The morphology of T cells loaded with microspheres was observed by Dapi staining. Flow cytometry was used to verify whether co-culture with microspheres influenced the ratio of CD4+ T cells to CD8+ T cells. 
RESULTS AND CONCLUSION: (1) Under the inverted microscope, the microspheres were highly dispersed and uniform in size. The diameter size met the injectable condition. Under scanning electron microscope, the microspheres formed porous structure after freeze-drying, and pores were uniformly distributed. The elastic modulus of the microspheres was (9.76±2.04) kPa. (2) CCK-8 assay and live dead cell staining results demonstrated that the tendency and activity of proliferation of MC3T3-E1 cells cultured in the two media had no difference. (3) Under optical microscope, co-culture with microspheres for 2 days did not cause CD3+ T cell activation, and did not interfere with the activation of CD3+ T cells. CD3+ T cells were distributed on the surface and pores of the microspheres. (4) Flow cytometry results showed that microspheres did not affect the ratio of CD4+ T cells to CD8+ T cells. (5) An injectable gelatin methacryloyl porous microsphere was prepared by microfluidic technology, which has good biocompatibility and does not affect cell. It is a kind of biomaterial with broad application prospect in tissue engineering. 

Key words: microfluidic, hydrogels, porous microspheres, injectable, biocompatibility, cell therapy, T cell, tissue engineering

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