制备负载细胞可注射微球及体外评价  

Preparation and in vitro evaluation of injectable microspheres loaded with cells

作　　者：何家辰 刘畅[1,2] 陈迟迟 施勤[1,2] He Jiachen;Liu Chang;Chen Chichi;Shi Qin(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)

机构地区：[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

出　　处：《中国组织工程研究》2022年第28期4483-4488,共6页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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。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 no

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 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]