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
姜明言 1田卫东 1谢利1
作者信息
- 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
- 折叠
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 24h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 7d内细胞存活率未明显降低(P＞0.05),CCK-8显示培养 4d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.
Abstract
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 inter-nal and external phase solutions and cell density,the morphology and size of the microcapsules were observed to get the best prepara-tion condition.Cell viability was detected through live/dead staining and CCK-8 assay,and the cytoskeleton was observed using immu-nofluorescence staining.Results:DPSCs were successfully isolated and cultured.Cell-laden microcapsule with alginate layer and car-boxymethylcellulose 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 stai-ning 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Key words
cell spheroids/coaxial-electrospray/dental pulp stem cells/dental pulp regeneration引用本文复制引用
基金项目
国家重点研发计划(2022YFA1104400)
国家自然科学基金(32271415)
出版年
2024
NETL
NSTL
万方数据