Fig 1: Long-Term, 3D Cultures of hAT2 Cells in Chemically Defined Conditions(A) Schematic diagram outlining our h3AC method.(B) A representative image of primary human alveolar type 2 (hAT2)-derived three-dimensional (3D) structures (h3ACs) from freshly isolated HTII-280+ cells at day 27 in culture (top) and with Lysotracker (bottom; red). P0, passage 0. Scale bars represent 2,000 µm (top) and 1000 µm (bottom).(C) Morphological heterotypic colony formation from isolated hAT2 cells in primary h3ACs (P0). Hematoxylin and eosin (H&E; left) and IF staining (right) for HTII-280 (green), pro-SFTPC (red), and DAPI (blue). Scale bar, 50 µm.(D) Quantification of the folded and cystic 3D structures in primary h3ACs (P0). Data presented are the mean ± SEM for three individual donor samples (n = 67 for donor 1, n = 50 for donor 2, and n = 50 for donor 3; n = total number of colonies scored).(E) IF images of primary h3ACs expressing HTII-280 (green), F-actin (white), CRB3 (red), and DAPI (blue). Scale bar, 50 µm.(F) IF images of primary h3ACs expressing HTII-280 (green), F-actin (white), CRB3 (red), SCRIB (red), and DAPI (blue). Scale bar, 50 µm.(G) Serial passage of h3ACs via single-cell dissociation at various time points depending on growth from three individual donors. Each point represents a single passage for n = 3 individual donor samples with more than three technical replicates.(H) Quantification of colony-forming efficiency for h3ACs at day 14 of culture up to nine total passages (colony-forming efficiency is defined as the number of colonies formed/number of cells plated per well as a percentage). Data are presented as mean ± SEM for three individual donor samples (n = 3 biological samples). Each point represents the average of three technical replicates calculated for each biological sample (except passage 8 [P8], where n = 2).(I) Flow cytometry analysis of Lysotracker (deep red) uptake in freshly isolated HTII-280+ cells and dissociated h3ACs at passage 6 (P6; 6 months in culture).(J) IF images of hAT1 cells, induced by 2D-plating of cultured h3ACs at passage 2. Pro-SFTPC (hAT2, white), AGER (hAT1, red), AQP5 (hAT1, green), and DAPI (blue). Scale bar, 50 µm.(K) Quantification of h3ACs expressing pro-SFTPC and TP63 over multiple passages (P0, passage 1 [P1], and P8). h3ACs were classified based on IF staining for pro-SFTPC (hAT2), TP63 (basal), or lack of both markers (pro-SFTPC- TP63-). The mean percentage of total colonies per well represented by each class of colony is shown. Data are presented as mean ± SEM at each passage (n = 131 for P0, n = 55 for P1, and n = 25 for P8). Three donor samples were used, except for P8 (where one donor sample was used). n = total number of colonies scored across passages.