Establishment and characterization of high-throughput culture platform for kidney organoids
10.3760/cma.j.cn441217-20220916-00924
- VernacularTitle:肾脏类器官高通量培养平台的建立及鉴定
- Author:
Le WANG
1
;
Xiaoqiang YANG
;
Yiming ZHOU
Author Information
1. 广东省重点实验室，中山大学孙逸仙纪念医院，广州 510120
- Keywords:
Kidney;
Organoids;
Induced pluripotent stem cells;
High-throughput culture
- From:
Chinese Journal of Nephrology
2023;39(3):200-208
- CountryChina
- Language:Chinese
-
Abstract:
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 1.0×10 7, approximately 7 renal organoids were obtained by the transwell protocol, while approximately 1 000 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.
