肾脏类器官高通量培养平台的建立及鉴定

Establishment and characterization of high-throughput culture platform for kidney organoids

目的建立及鉴定诱导多能干细胞分化肾脏类器官的高通量培养平台。方法选择人尿源诱导多能干细胞, 以适合的细胞密度接种铺板, 第1~6天在CHIR99021/成纤维细胞生长因子9/肝素等小分子化合物诱导下分化;在第7天将适合密度的细胞消化后重悬, 加入到96孔V型细胞培养板进行3D悬浮培养24 h, 待细胞形成球体之后, 继续加入成纤维细胞生长因子9/肝素诱导分化。第24天诱导分化成熟, 与已报道的分化方案(Transwell方案)所获得的肾脏类器官进行免疫荧光和透射电镜比较。结果两种方案均可成功分化出肾脏类器官。免疫荧光结果显示肾脏主要细胞标志物LTL、GATA-3、Synaptopodin均有表达, 形成成熟的肾脏类器官。透射电镜结果显示肾脏类器官形成许多相互交错穿插、分布均匀及排列整齐的足突, 符合肾脏足细胞特有细胞结构的特征。在1.0×107相同中间中胚层细胞数下, Transwell方案获得约7个肾脏类器官, 而高通量培养方案获得约1 000个肾脏类器官。结论肾脏类器官高通量培养平台成功建立, 可获得大量的成熟且结构和功能完善的肾脏类器官, 极大地提高了肾脏类器官的分化效率。

Objective To establish and identify a high-throughput culture platform for induced pluripotent stem cells to differentiated kidney organoids.Methods Human urine-derived induced pluripotent stem cells were selected and plated at a suitable cell density,and differentiated using small molecule compounds such as CHIR99021/fibroblast growth factor 9/heparin during day 1-6.On day 7,cells with appropriate density were digested and resuspended,than added to a 96-well 3D culture plate for 24 hours.After the cells formed spheroids,fibroblast growth factor 9 and heparin were added to induce differentiation till day 24.The immunofluorescence and transmission electron microscopy were used to compare the differences of kidney organoids obtained by the reported differentiation protocol(transwell protocol)method and high-throughput culture platform.Results Kidney organoids were successfully differentiated by two protocols.Immunofluorescence results showed that LTL,GATA-3,and synaptopodin,which were major kidney cell markers,were all expressed,and mature renal organoids were formed.The results of transmission electron microscopy showed that the kidney organoids successfully developed foot processes,the unique cellular feature of the glomerular podocytes,which were evenly distributed and neatly interspersed with each other.At the same intermediate mesodermal cell count of 1.0×107,approximately 7 renal organoids were obtained by the transwell protocol,while approximately 1000 renal organoids were obtained by the high-throughput culture platform.Conclusion A high-throughput culture platform for kidney organoids is successfully established,and a large amount of mature kidney organoids with complete structure and function can be obtained.The differentiation efficiency of kidney organoids is greatly improved.