Proliferative Activity and Changes in the Relative Proportion of TI and TII Populations in Cultured Primary Epithelial Cells, Isolated from Mouse Lung
Mouse lung tissue was digested with dispase and collagenase enzymes to dissociate cells and primary lung epithelial (PLE) cells were isolated by removing leukocytes, erythroid cells, endothelial cells and fibroblasts, using a magnetic-bead based negative selection process. Purified PLE cells were cultured on fibronectin coated plastic dishes. Proliferative activity in PLE cell in culture was monitored by using time lapse live imaging technique. Proliferative activity of PLE cells started after 3 days in culture and peaked on day 4 to 5 when about 11% of the cells in culture were seen to divide. Proliferation activity ceased after day 6 to 7. There was a marked change in the morphology of the cells as the cultured cells became flattened and significantly larger in size. Type I and type II PLE cells were enumerated in culture by flow cytometry based upon staining with anti-podoplanin antibody (type I cells) or CD74 antibody (type II cells). In freshly isolated PLE cells, type I and type II epithelial cells accounted for about 20% each, bulk of the remaining cells being double negative. The proportion of type I cells rapidly increased in culture and was above 80% by day 4 of the culture. Proportion of cells expressing B7.1 and ICAM-1 molecules involved in antigen presentation increased but the MHC class II+ cells declined. These results show that PLE cells isolated by negative selection show a transient proliferative activity in culture become predominantly type I epithelial cells and retain the expression of some important antigen presentation markers.