Journal: Respiratory Research

Article Title: Optimized culture of primary human alveolar type II cell–derived 3D organoids from fibrotic lung tissue with phenotypic and metabolic profiling

doi: 10.1186/s12931-026-03610-9

Figure Lengend Snippet: Isolation of AT-II and AT-I epithelial cells by magnetic bead sorting. A . Schematics of sorting-based isolation. Cohort of this study included 63 lung tissues derived from N = 15 IPF and N = 26 secondary pulmonary fibrosis patients in addition to N = 22 tumor-distant, pathologically inconspicuous control tissues in total. Per each experiment or technique, defined numbers of N from fibrotic or “healthy” from this cohort were utilized as indicated in corresponding figure legends. Primary AT-II cells were isolated from human lung explants or partial tumor resections. Following magnetic bead sorting by HT-II-280, AT-II cells were cultured in matrigel domes to form alveolospheres. B . Exemplary display of cytospins of unsorted whole lung cell population vs. freshly MACS sorted cells using anti-HT-II-280 mouse IgM reveal the expression of AT-II-specific markers. HT-II-280 (red) and proSP-C (green) localize to AT-II cells. Images of unsorted and sorted cells in cytospins are derived from the same patient. Cell nuclei were stained in blue using DAPI staining. Representative picture of 5 patient tissues for sorted fraction ( N = 2 from healthy tumor-distant control tissue, N = 3 from fibrotic end-stage lungs) and of 4 patient tissues for unsorted fraction ( N = 2 from healthy tumor-distant control tissue, N = 2 from fibrotic end-stage lungs). Scale bars = 20 µm. C . Quantification of HT-II-280 + proSP-C + cells/total to DAPI + cytospinned cells following directly after HT-II-280 MACS indicate an overlapping population of both AT-II cell markers. Individual data points display the mean of 5 images recorded per each patient tissue. One-way ANOVA (***, p = 0.0079) shows statistical significance between means of single- or double positive cell populations before and after sorting which is further demonstrated in Tukey ‘s multiple comparison test indicated in graph. Sorted fraction displays insignificant differences (ns, p = 0.0974 for HT-II-280 + vs. HT-II-280 + proSP-C + cells; ns, p = 0.0631 for proSP-C + vs. HT-II-280 + proSP-C + cells). However, HT-II-280 + are strongly enriched after sorting (****, p < 0.0001) alongside proSP-C + cells (**, p = 0.0091) or double-positive cells (***, p = 0.00411). Representative picture of 6 tissues ( N = 3 from healthy tumor-distant control tissue, N = 3 from fibrotic explants). D . Exemplary flow cytometry measurement before and directly after MACS-based sorting illustrates strong enrichment of HT-II-280 + cells directly post-sorting. This indicates that a relevant fraction of surface epitopes remains accessible despite prior bead binding, although potential epitope competition or steric hindrance should be considered when interpreting absolute signal intensities. Majority of sorted cells further express proSP-C. Gating was performed to exclude debris and focus on single cells, see Supplementary Fig. 1. Representative data of total N = 7 ( N = 3 healthy tissue donors and N = 4 fibrotic explants). E . Cytospins of unsorted whole lung cell population vs. freshly MACSed cells using anti-HT-I-56 ms IgG highlights the suitability to also isolate AT-I cells from the same tissue with similar approach. Cell nuclei were stained in blue using DAPI staining. Representative picture of 4 tissues ( N = 2 from healthy tumor-distant control tissue, N = 2 from fibrotic explants). Scale bar = 20 µm. F . Quantification of HT-I-56 expression in cytospinned cells. Individual data points display the mean of 5 images recorded per each patient tissue. Numbers of HT-I-56-expressing cells relative to DAPI staining. Paired t-test showed significant enrichment of HT-I-56. + following sorting (***, p = 0.00452). Representative picture of 5 tissues ( N = 2 from healthy tumor-distant control tissue, N = 3 from fibrotic explants)

Article Snippet: For AT-I isolation, the primary antibody was replaced by mouse IgG anti-HT-I-56 (Terrace Biotech, Cat. No. TB-29AHT1-56) at the same dilution.

Techniques: Isolation, Derivative Assay, Control, Cell Culture, Expressing, Staining, Comparison, Flow Cytometry, Binding Assay