基于核壳结构微囊构建三维牙髓细胞球及初步体外研究  

In vitro study of core-shell microcapsules for dental pulp stem cell spheroid formation

作　　者：姜明言 田卫东[1] 谢利 JIANG Mingyan;TIAN Weidong;XIE Li(State Key Laboratory of Oral Diseases,National Clinical Research Center for Oral Diseases,Engineering Research Center of Oral Translational Medicine,Ministry of Education,National Engineering Laboratory for Oral Regenerative Medicine,Department of Oral and Maxillofacial Surgery,West China Hospital of Stomatology,Sichuan University,Chengdu 610041,China)

机构地区：[1]口腔疾病防治全国重点实验室,国家口腔疾病临床医学研究中心,口腔转化医学教育部工程研究中心,口腔再生医学国家地方联合工程实验室,四川大学华西口腔医院创伤与整形外科,四川成都610041

出　　处：《口腔生物医学》2024年第2期75-81,90,共8页Oral Biomedicine

基　　金：国家重点研发计划(2022YFA1104400);国家自然科学基金(32271415)。

摘　　要：目的:利用同轴静电液滴技术设计并制备一种用于牙髓干细胞(DPSCs)三维细胞球体构建、培养的核壳微囊。方法:获取临床收集DPSCs并通过流式细胞仪进行细胞鉴定;构建核壳微囊,调节内外相溶液浓度、流速和细胞密度等参数,对比微囊形貌及尺寸筛选最佳制备条件;活/死染色和CCK-8实验检测微囊内的细胞球的细胞活性,免疫荧光染色观察细胞骨架。结果:成功分离培养DPSCs,构建以海藻酸钠(Alg)为壳层和羧甲基纤维素(CMC)为核层的载细胞微囊。当细胞密度为1×108个/mL时,微囊内的DPSCs在24 h内能自发形成直径为(101.93±10.11)μm的单一球体。活/死染色显示培养7 d内细胞存活率未明显降低(P>0.05),CCK-8显示培养4 d时细胞增殖率增加至(114.91±7.70)%(P<0.05),7 d时增长至(169.05±5.29)%(P<0.001)。细胞在微囊内形成致密结构。结论:该方法制备的核壳微囊可以促进DPSCs形成三维细胞球并长期存活。Objective:To develop a novel strategy for the construction and cultivation of three-dimensional cell spheroids from dental pulp stem cells(DPSCs)with coaxial-electrospray method.Methods:DPSCs were isolated from clinically collected teeth and then identified through flow cytometry.Core-shell microcapsules were constructed.By adjusting the concentration and flow rates of internal and external phase solutions and cell density,the morphology and size of the microcapsules were observed to get the best preparation condition.Cell viability was detected through live/dead staining and CCK-8 assay,and the cytoskeleton was observed using immunofluorescence staining.Results:DPSCs were successfully isolated and cultured.Cell-laden microcapsule with alginate layer and carboxymethylcellulose core structure was fabricated.When the cell density within microcapsules reached 1×108 cells/mL,the DPSCs within a single microcapsule formed a spheroid spontaneously with a diameter of(101.93±10.11)μm within 24 hours.Live/dead staining showed the cell survival rate didnot decrease within 7 days(P>0.05).CCK-8 assay revealed an increase in cell proliferation rate to(114.91±7.70)%after 4 days of culture(P<0.05),which further grew to(169.05±5.29)%at 7 days(P<0.001).Cells formed dense structures within spheroids.Conclusions:These core-shell microcapsules could promote the formation of DPSCs cell spheroids and maintain their long-term survival.

关 键 词：细胞球状体 同轴静电液滴 牙髓干细胞 牙髓再生 

分 类 号：R780.2[医药卫生—口腔医学]