摘要
背景:相较于2D培养技术,3D培养技术能够更准确地模拟细胞自然生长的体内环境,成为基础科学研究和转化医学领域的关键焦点。目的:通过3D培养技术构建预血管化的成骨类器官。方法:从同一人体脂肪组织中分别分离提取血管基质成分、脂肪间充质干细胞,通过流式细胞学鉴定细胞组分。将血管基质成分、脂肪间充质干细胞分别接种于96孔U型板内,培养3 d后血管基质成分自组装成型、脂肪间充质干细胞成球,培养第14天,通过苏木精-伊红染色与细胞骨架染色观察球体内部结构和细胞形态;培养0,7,14,21 d,Presto Blue实验检测细胞活性;培养3 d后分别进行成骨、成软骨与成脂诱导分化,诱导14 d后,茜素红染色观察成骨分化,阿利新蓝染色观察成软骨分化,油红O染色观察成脂分化,免疫荧光染色共定位成骨诱导后CD31(内皮细胞标志物)、RUNX2(早期成骨标志物)、CD44(间充质干细胞标志物)、Ⅰ型胶原(晚期成骨标志物)、过氧化物酶体增殖物激活受体γ(成脂标志物)的表达,qRT-PCR检测成骨诱导后RUNX2、骨桥蛋白、Ⅰ型胶原、Sp7转录因子、CD31和血管内皮生长因子mRNA表达。结果与结论:①流式细胞分析表明,血管基质成分包含脂肪间充质干细胞和内皮细胞等多种细胞类型,脂肪间充质干细胞球主要由脂肪间充质干细胞构成;②苏木精-伊红染色及细胞骨架染色显示,相对于脂肪间充质干细胞球,血管基质成分类器官结构更贴近天然组织,细胞排列有序,细胞外基质更丰富;细胞活性测试显示,脂肪间充质干细胞球培养7 d的细胞活性高于血管基质成分类器官(P<0.05),培养21 d的细胞活性低于血管基质成分类器官(P<0.05),说明血管基质成分类器官的细胞活性持续时间更长;③相较于脂肪间充质干细胞球,血管基质成分类器官具有更优的骨、软骨和脂肪形成能力;多色免疫荧光共定位显示,血管基质成分类器官中血管网络和成骨标志物表达丰富,细胞间和细胞-基质相互作用复杂,脂肪间充质干细胞球结构单一且缺乏内皮细胞基因表达;qRT-PCR检测结果显示,血管基质成分类器官中RUNX2、骨桥蛋白、Ⅰ型胶原、Sp7转录因子、CD31和血管内皮生长因子mRNA表达均高于脂肪间充质干细胞球(P<0.05);④结果表明,血管基质成分类器官具备复杂的生理结构和更强的血管形成和骨形成能力,以及更持久的细胞活性。
BACKGROUND:Compared with two-dimensional culture techniques,three-dimensional culture technology can more accurately simulate the in vivo environment of natural cell growth,which becomes a key focus in the fields of basic scientific research and translational medicine.OBJECTIVE:To construct prevascularized osteogenic organoids through three-dimensional culturing techniques.METHODS:Stromal vascular fraction(SVF)and adipose mesenchymal stem cells were isolated and extracted from the same human adipose tissue,and the cell components were identified by flow cytometry.The SVF and adipose mesenchymal stem cells were inoculated into 96-well U-plates.After 3 days of culture,the SVF was self-assembled and the adipose mesenchymal stem cells were formed into spheres.On the 14th day of culture,the internal structure of the spheres and cell morphology were observed by hematoxylin-eosin staining and cytoskeletal staining.Viability of the cells was detected by Presto Blue assay on days 0,7,14,and 21 of culture.Osteogenic,chondrogenic,and lipogenic differentiation were induced after 3 days of culture.After 14 days of induction,alizarin red staining was used to observe osteogenic differentiation;alizarin blue staining to observe chondrogenic differentiation;oil red O staining to observe lipogenic differentiation;immunofluorescence staining to co-localize CD31(endothelial cell marker),RUNX2(early osteogenic marker),CD44(mesenchymal stem cell marker),type I collagen(late-stage osteogenic marker),and peroxisome proliferator-activated receptorγ(lipogenic markers);and qRT-PCR to detect the mRNA expression of RUNX2,osteopontin,type I collagen,Sp7 transcription factor,CD31,and vascular endothelial growth factor.RESULTS AND CONCLUSION:(1)Flow cytometry results revealed that the SVF contained various cell types including adipose mesenchymal stem cells and endothelial cells,whereas adipose mesenchymal stem cell spheroids were primarily composed of adipose mesenchymal stem cells.(2)Hematoxylin-eosin staining with cytoskeleton staining indicated that compared with adipose mesenchymal stem cell spheroids,SVF organoids more closely mimicked natural tissue structures with orderly arranged cells and richer extracellular matrix.Cell viability tests showed that adipose mesenchymal stem cell spheroids had higher activity on day 7,but SVF organoids surpassed the cells by day 21(P<0.05),exhibiting longer-lasting cell viability.(3)In tri-lineage differentiation experiments,SVF organoids demonstrated superior osteogenic and chondrogenic potential compared with adipose mesenchymal stem cell spheroids.Multicolor immunofluorescence colocalization revealed rich vascular networks and pronounced osteogenic marker expression in SVF organoids,with complex cell-to-cell and cell-to-matrix interactions,whereas adipose mesenchymal stem cell spheroids showed a simpler structure with a lack of endothelial gene expression.qRTPCR results indicated significantly higher expression of RUNX2,osteopontin,type I collagen,Sp7 transcription factor,CD31,and vascular endothelial growth factor in SVF organoids compared with adipose mesenchymal stem cell spheroids(P<0.05).Overall,the findings suggest that SVF organoids possess complex physiological structures with enhanced capabilities in vascular and bone formation,as well as sustained cell viability.
作者
吴佳洲
钱陶
刘泽贤
武艳斌
何莹
李亚洲
彭江
Wu Jiazhou;Qian Tao;Liu Zexian;Wu Yanbin;He Ying;Li Yazhou;Peng Jiang(Department of Orthopedics,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Institute of Orthopedics,Fourth Medical Center,Chinese PLA General Hospital,Beijing 100853,China)
出处
《中国组织工程研究》
北大核心
2026年第11期2681-2690,共10页
Chinese Journal of Tissue Engineering Research
基金
国家重点研发计划课题项目(2024YFA1108605),项目负责人:彭江。
关键词
三维培养
血管基质成分
脂肪间充质干细胞
内皮细胞
类器官
成骨分化
血管生成
工程化材料
three-dimensional culture
stromal vascular fraction
adipose mesenchymal stem cells
endothelial cells
organoids
osteogenic differentiation
angiogenesis
engineered materials
