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Establishment of a three-dimensional organoid culture system for mouse type 2 alveolar epithelial cells

WEI Juan^1,2, XU Chu-Fan³, ZHU Xiao-Yan⁴, LIU Yu-Jian^1,*

¹School of Kinesiology, The Key Laboratory of Exercise and Health Sciences of Ministry of Education, Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, Shanghai University of Sport, Shanghai 200438, China；²School of Sports and Health, Nanjing University of Sport, Nanjing 210014, China；³Department of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China；⁴Department of Physiology, Navy Medical University, Shanghai 200433, China

Abstract

The purpose of this study was to establish a three-dimensional (3D) organoid culture system for type 2 alveolar epithelial (AT2) cells in mice. AT2 cells were isolated from ICR mouse lung and purified by enzymatic digestion and MicroBeads sorting. The purity of AT2 cells was determined by immunofluorescence (IF) staining using an antibody against proSPC. The AT2 differentiation was examined by IF staining with proSPC/HopX and proSPC/T1α antibodies, and proliferation of AT2 cells was assessed by EdU incorporation assays after two-dimensional (2D) culture for 8 days. In addition, AT2 cells were co-cultured with mouse lung fibroblasts (Mlg) in three-dimensional (3D) culture system. After 13 days of co-culture, the organoids were fixed in 2% paraformaldehyde for histological analysis and IF staining. The results showed that the purity of the AT2 cells was over 95%, as assessed by proSPC staining. 2D cultured AT2 cells were negative for EdU staining, which indicates that no proliferation occurs. proSPC expression was gradually disappeared, whereas T1α and HopX expression was gradually increased after 3, 5 and 8 days of culture. In 3D culture system, the alveolar organoids were formed after co-culturing AT2 cells with Mlg for 4 days. Histological analysis showed that alveolar organoids displayed a hollow morphology. proSPC was highly expressed in the peripheral cells, whereas type 1 alveolar epithelial (AT1) cells transdifferentiated from AT2 cells expressing HopX were mainly located in the interior of organoid bodies after 13 days. Some of the proSPC-positive AT2 cells located in the outer circle of alveolar organoids were stained positive for both proSPC and EdU, indicating that the AT2 cells in the alveolar organoids were proliferative. These results showed that the 3D organoid culture system of mouse AT2 cells was successfully established.

Key words: AT2; AT1; mouse lung fibroblasts; differentiation; proliferation; organoids

Received: 　　Accepted:

Corresponding author: 刘宇健　　E-mail: liuyujian@sus.edu.cn

DOI: 10.13294/j.aps.2022.0060

Citing This Article：

WEI Juan, XU Chu-Fan, ZHU Xiao-Yan, LIU Yu-Jian. Establishment of a three-dimensional organoid culture system for mouse type 2 alveolar epithelial cells. Acta Physiol Sin 2022; 74 (4): 585-595 (in Chinese with English abstract).