Isolation, identification and construction of three-dimensional culture system of mouse bronchoalveolar stem cells

ObjectiveTo explore the method of isolation, identification and three-dimensional culture system of bronchioalveolar stem cells (BASCSs) from C57BL6 mouse in vitro, so as to lay an experimental foundation for further study of related characteristics of BASCs.

MethodsThe lung tissue of adult male C57BL6 mice was completely isolated, lavaged by 1×PBS, digested by Elastase solution perfused through the trachea.The lung tissue was cut up with a sharp instrument, the cells were better dispersed using DNA enzymes, and the undigested tissue was removed with 75 μm sieve.The cells suspensions were stained using the fluorescent labeled CD31, CD34, CD45, SCA-1 and EpCAM antibody.The CD31^-CD34^-CD45^-SCA-1⁺ EpCAM⁺ cells were sorted out using flow cytometry, and mixed with Mlg2908 cells (mouse lung fibroblast cell line) at the ratio of (1×10³):(1×10⁶) to construct a three-dimensional culture system based on the Matrigel as matrix.

ResultsThe lung tissue was obstained by Elastase digesting, the 1.6×10⁷ to 1.8×10⁷ nucleated cells were obtained from a mouse.The results of flow cytometry showed that the CD34^- CD45^- SCA-1⁺ EpCAM⁺ cells accounted for about 22% of EpCAM⁺ cells.BASCSs, Mlg2908 cells and Matrigel were mixed and cultured.After 4 d, the clone was observed.With the extension of culture time, the number of clones decreased gradually.After 8 days, most of the clones were 50 μm in diameter, differentiated in morphology, and could sustain for 14 d.

ConclusionsA simple and efficient method for isolation, identification and three-dimensional culture of mouse BASCSs is successfully established.