Human Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine

Human Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Human Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine Lara-Jasmin Schröder, Julia Rückoldt, Stephanie Schubert, Lars Knudsen, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7913465/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Respiratory Research → Version 1 posted 9 You are reading this latest preprint version Abstract Background Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II identity, enable cryopreservation, and capture disease-specific metabolic alterations. Methods HT-II-280 + AT-II cells were isolated by magnetic bead sorting from 62 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 21 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II identity was verified by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability. Metabolic profiling was performed using extracellular flux assays. Results AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Interestingly, primary tissue-derived lung organoids display heterogeneous morphologies and sizes, particularly in fibrotic-derived cultures indicated by histology and microcomputed tomography. Culturing conditions were optimized to avoid differentiation towards AT-I cells or aberrant basaloid cells. Lineage fidelity was preserved across passages, with stable expression of proSP-C, HT-II-280, and pronounced presence of lamellar bodies. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, highlighting possibility for on demand long-term storage. Fibrotic organoids exhibited metabolic reprogramming illustrated by a pronounced glycolytic shift with increased ATP production. Conclusion We established a robust and reproducible cell-line-free 3D platform from primary human AT-II cells of end-stage ILD lungs to generate personalized lung organoids. These organoids retain AT-II identity across passages, remain viable after cryostorage, and recapitulate patient-specific metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting possible mitochondrial dysfunction and high energy demand. This reproducible scalable model provides a transferable resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for precision medicine. alveolar type II cells lung epithelium lung organoids alveolosphere Idiopathic pulmonary fibrosis human lung explant fibrotic end-stage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease (ILD) characterized by excessive extracellular matrix (ECM) deposition, loss of alveolar structure, and respiratory failure. The disease primarily affects older adults and has a poor prognosis, with a median survival of only 3–5 years after diagnosis [ 1 ] . Current anti-fibrotic therapies can slow progression but neither halt nor reverse fibrosis [ 2 ] . The lack of curative options highlights the urgent need for human-derived disease models that recapitulate IPF pathogenesis [ 3 ] . Alveolar type II (AT-II) epithelial cells play a central role in alveolar homeostasis and fibrotic remodeling. These cuboidal surfactant-producing cells constitute ~ 5% of the alveolar surface, secrete surfactant, regulate immune responses, and act as progenitors for alveolar type I (AT-I) cells [ 4 ] . Under normal conditions, AT-II cells self-renew and transdifferentiate into AT-I cells after injury [ 4 ] . In IPF, however, AT-II cells undergo apoptosis, senescence, or aberrant activation, contributing to chronic epithelial damage and fibrosis [ 5 ] . In addition to these processes, AT-II cells in fibrotic lungs can give rise to aberrant basaloid cells, a transitional epithelial state observed in distal fibrotic regions of IPF [ 6 – 8 ] . Recent studies, including single-cell transcriptomic analyses, suggest that matrix stiffening and altered mechanical cues drive this basaloid differentiation program [ 6 ] . Such stiffness-induced epithelial transitions have been demonstrated in vitro and in vivo, where direct contact with rigid substrates promotes loss of AT-II identity and activation of basal markers [ 6 , 8 – 9 ] . They may further sustain the fibrotic niche by secreting profibrotic cytokines, undergoing epithelial–mesenchymal transition (EMT), and serving as progenitors of myofibroblasts [ 10 ] . AT-II cells can be identified by lineage-defining markers such as surfactant proteins (SFTPC, SFTPB), ABCA3 [ 11 ] , LAMP3 [ 12 ] , NKX2-1/TTF-1, and the surface marker HT-II-280 used for human isolation [ 13 – 14 ] . Morphologically, the presence of lamellar bodies is a characteristic hallmark of AT-II cells [ 15 – 16 ] . AT-I cells, in contrast, cover ~ 95% of the alveolar surface and express RAGE/ AGER, HT-I-56, and HOPX 17–18] . In addition to AT-II–derived fibroblast-like states, injured AT-I cells in IPF adopt dysfunctional/senescent programs that secrete pro-fibrotic mediators (e.g., TGF-β, CTGF, WNT ligands), thereby activating fibroblasts and amplifying ECM deposition; AT-I–restricted regulators such as AGER (RAGE) and caveolin-1 further modulate this crosstalk, and their dysregulation has been linked to heightened fibrosis [ 19 – 22 ] . Recent studies showed that IPF-derived AT-II cells undergo metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis, a “Warburg-like” phenotype. This is linked to mitochondrial dysfunction, elevated glycolytic enzyme expression (LDHA, PDK1), lactate accumulation, and suppression of pyruvate dehydrogenase via the PDK1–HIF-1α axis [ 23 – 25 ] . Such energetic shifts are thought to drive fibrotic remodeling and represent potential therapeutic targets. Organoids—self-organizing 3D epithelial culture systems—have become valuable models for disease research. Pulmonary organoids derived from fetal, adult, or induced pluripotent stem cell (iPSC) sources can recapitulate alveolar architecture and function [ 26 – 28 ] . They allow long-term expansion, passaging, and cryopreservation while maintaining lineage identity, enabling applications in disease modeling, drug testing, and personalized medicine [ 29 – 30 ] . Several lung organoid models reproduced features of fibrosis, epithelial plasticity, and anti-fibrotic responses [ 31 – 32 ] . Compared to 2D cultures, organoids better preserve epithelial polarity, surfactant secretion, and progenitor functions. However, robust primary human AT-II organoid models from end-stage fibrotic lungs are still lacking. Most existing models rely on murine or iPSC-derived epithelium and do not reflect the native metabolic or regenerative state of diseased AT-II cells. Moreover, current systems often lack reproducibility across laboratories and limited scalability, restricting their broader translational use. A reproducible, feeder-free human AT-II model that allows cryopreservation and biobanking would therefore be highly valuable for precision medicine and cross-center validation. Here, we establish a defined feeder-free 3D culture system for primary human HT-II-280 + AT-II cells isolated from both healthy and fibrotic (IPF and secondary fibrosis) lung tissue adapted from previous work by Konishi et al. [ 33 ] and Katsura et al. [ 27 ] . We aimed to (i) maintain AT-II identity across passages using canonical markers (HT-II-280, proSP-C, lamellar bodies), (ii) assess cryopreservation and potential for organoid biobanking, and (iii) evaluate metabolic differences between healthy and fibrotic AT-II organoids using extracellular flux analysis. We hypothesized that fibrotic AT-II cells exhibit enhanced glycolytic metabolism compared to controls, reflecting disease-specific metabolic reprogramming. By providing a robust and transferable protocol, our study delivers a reproducible human lung organoid resource that supports mechanistic and translational research in ILD. Methods Patient cohort and tissue collection For this study, we analysed 62 lung tissues (22 females, 40 males; average age 60 ± 12 years), including 41 fibrotic explants (15 IPF, 26 secondary pulmonary fibrosis) and 21 tumor-distant “healthy” controls. Tissues were used either for one or multiple investigations. Donors provided written informed consent, and the study was approved by the Hannover Medical School ethics committee (8867_BO_K_2020, 10194_BO_K_2022). Patient demographics and tissue utilization are summarized in Table 1. Isolation of primary alveolar epithelial cells Primary human alveolar type II (AT-II) and type I (AT-I) cells were isolated from freshly explanted fibrotic or tumor-distant “healthy” control lung tissue by magnetic-activated cell sorting (MACS) using the AT-II marker HT-II-280 and the AT-I marker HT-I-56, respectively [ 13 , 17 , 27 , 33 ] . All steps were performed on ice or at 4°C unless otherwise specified and all buffers and consumables were pre-cooled. Peripheral lung tissue was dissected free of pleura, bronchi, and large vessels, mechanically minced, and enzymatically digested (Miltenyi Biotec, gentleMACS and No. 130-110-201 Multi Tissue Dissociation Kit 1) [Miltenyi Biotec, Cat. No. 130-110-201]). After sequential filtration (100/70 µm), centrifugation (450 g, 7 min, 4°C), and erythrocyte lysis with ACK lysis buffer (Thermo Fisher Scientific/Gibco, Cat. No. A10492-01) followed by sequential filtration (40 µm), cells were washed and resuspended in MACS buffer (1% BSA, 2 mM EDTA in PBS). Following Fc receptor blockade, (10 µL/sample; Miltenyi Biotec) for 15 min at 4°C, cells were incubated with 5 µL mouse IgM anti-HT-II-280 antibody (Terrace Biotech, Cat. No. TB-27AHT2-280; 1:50 in MACS buffer) for 1 h at 4°C with gentle rotation (60 rpm). 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. Cells were washed with 1.5 mL MACS buffer, centrifuged, and resuspended in 2 mL MACS buffer containing anti-mouse IgM or anti-mouse IgG MicroBeads (Miltenyi Biotec; 20 µL beads per 10 7 cells, diluted 1:10 in MACS buffer). Bead incubation was carried out for 30 min at 4°C with gentle rotation. After washing, suspensions were applied to pre-equilibrated LS columns in an OctoMACS separator. Negative fractions were collected as flow-through, while positive fractions were eluted after column removal from the magnet. Both fractions were pelleted, resuspended in MACS buffer, and used for counting or downstream applications. MACS was selected over FACS to reduce shear stress and preserve viability for organoid culture. Both positive and negative fractions were pelleted (450 g, 7 min, 4°C) and resuspended in 200 µL MACS buffer for counting or immediate downstream applications. 3D organoid culture MACS-isolated HT-II-280 + AT-II cells were pelleted (450 g, 7 min, 4°C) and resuspended in serum-free, feeder-free (SFFF) medium based on Advanced DMEM/F-12 (Gibco, Cat 12634028) supplemented with a variety of proliferation enhancers e.g. GSK3β-Inhibitor CHIR99021 and AT-I cell differentiation inhibitors such as ROCK-Inhibitor Y-27632, TGF-β-Receptor I (ALK5)-Inhibitor SB431542 and p38 MAPK-Inhibitor BIRB796 (see Supplementary Table S1 ). SFFF medium was adapted from Konishi et al. [ 33 ] using the same components but with modified concentrations to optimize maintenance of AT-II cell phenotype and increased proliferation. Cells in SFFF medium were mixed with Corning® Matrigel® Growth Factor Reduced Basement Membrane Matrix (Cat. No. 356231) at a 5:8 ratio (cell suspension:matrigel) and seeded as 100–130 µL domes in 24-well plates (60,000 cells/dome) in initial passage (P0). After polymerization (20–30 min, 37°C), 500 µL SFFF medium was added per well. For the first 4–5 days, IL-1β was added to enhance alveolosphere formation. Culture medium also contained EGF and FGF-10 to support Wnt and ERK/AKT pathway activation, essential for AT-II expansion. Organoids were grown for up to 16 days and passaged up to P3, with medium exchanged every 2–3 days. To generate single-cell suspensions for passaging of AT-II cells, organoids were segregated using TrypLE™ Select Enzyme (Gibco, Cat. No. 12563011). Per matrigel dome, 500 µL TrypLE™ Select Enzyme were utilized to firstly mechanically disrupt domes and incubate them at 37°C for 15–30 min with regard to organoid sizes and numbers. Suspensions were again mechanically disrupted, washed 3x in PBS, and centrifuged at 500 g for 5 min before further processing. Technical note : For all histological investigations and especially for flow cytometry labeling, we strongly recommend the utilization of Cell Recovery Solution (Corning, Cat. No.: 354253) before further processing. 300 µL Cell Recovery Solution were added to the domes to dissolve organoids from matrigel for more surface efficient stainings (e.g. such as for HT-II-280 labeling) and minimization on unlabeled cells in FACS to to residual matrigel. Organoids free of matrigel can further undergo immediate trypsination. Cryopreservation For cryopreservation, organoids were mechanically and enzymatically dissociated as described above. Up to 500,000 single cells were resuspended in 1 ml cryomedium (70% FBS, 20% SFFF medium, 10% DMSO, added dropwise). Cryomedium was incubated with AT-II for 20min at RT to allow for proper diffusion of DMSO. Cryovials were placed in a Mr. Frosty™ container at − 80°C and transferred after 24 h to − 150°C for long-term storage. To assess safety and viability, samples were stored ≥ 28 days at − 150°C. For recovery, cells were thawed in a 37°C water bath for 2 min, diluted in 14 ml Advanced DMEM/F-12 at room temperature, incubated for 10 min, centrifuged (7 min, 450 g), and directly reseeded in Matrigel at a 5:8 ratio. In-depth characterization of alveolar organoids To confirm the identity and purity of alveolar organoids over multiple passages and to evaluate cryostorage potential, a multitude of techniques was applied including conventional histology, microcomputed tomography, transmission electron microscopy, image analysis, immunofluorescence & flow cytometry as well as viability assays. For the sake of space, these techniques are described in detail in the supplement. Extracellular flux assays To assess the metabolic properties of fibrotic vs control organoids, glycolytic capacity and ATP production of fibrotic and healthy tissue-derived AT-II cells measured by extracellular flux assays using the Agilent Seahorse XF HS Mini Analyzer S7852A platform (Agilent, Santa Clara, CA). Organoids from P0-P1 were separated to single-cell suspension using trypsination as previously described in method section . Next, 12,000 cells from P1-P2 were plated 10–12 d prior to experiments in each well of XFp PDL Miniplates (Agilent, Cat. No.: 03722 − 100) in matrigel domes with 50 µl total volume as suggested by Ludikhuize et al . [ 34 ] and provided with SFFF media. By day of experiment, each well therefore roughly contains 85,000-120,000 cells as organoids. Samples were prepared in duplets. Cartridges were prepared corresponding to the manufacturer’s instructions for Seahorse XFp Glycolysis Stress Test Kit (Agilent, Cat. No.: 103017-100) and Seahorse XFp Real-Time ATP Rate Assay Kit (Agilent, Cat. No.: 103591-100). On the day of experiment, assay media were freshly prepared. SFFF was aspirated from matrigel domes exhibiting normal sized alveolospheres after cultivation miniplates and exchanged with 100 µl assay media 45 min prior to measurement, resulting in a total volume of 150 µl per well. Of note, after serial titration experiments for Seahorse XFp Glycolysis Stress Test Kit , we increased the concentrations of glucose to 15 mM, oligomycin to 5 µM and 2-DG to 200 mM per well, each resuspended in assay medium. Each injection port contained 20 µl volume. We measured 3 cycles each basal curve, glycolysis, and glycolytic capacity followed by 5 cycles of glycolytic reserve asessement. Similarly, for Seahorse XFp Real-Time ATP Rate Assay Kit , oligomycin (5µM) and Retenone + Antimycin A (2µM) per well were utilized in higher concentration than recommended from Agilent for measurement of 2D cell culture and with a volume of 20 µl per injection port. These concentrations are roughly in line with concentration utilized to measure extracellular flux of intestinal organoids by Ludikhuize et al . [ 34 ] . ATP production were assessed by 3 cycles each basal ECAR and OCR, inhibition of mitochondrial ATP synthesis, and, finally, mitochondrial-associated acidification. Following measurements, organoids were collected from matrigel using Cell Recovery Solution (Corning, Cat. No.: 354253) and harvested for DNA isolation using QIAamp DNA micro kit (Qiagen, Cat. No.:56304) and samples were measured by Nanodrop to enable a normalization of experimental values relative to approximated number of cells. Additionally, BrdU assays (Cell Signaling Technology, Cat. No.: 6813S) were performed to confirm unaltered cell proliferation and viability post-extracellular flux assays (data not shown). Statistical analysis All statistical analyses were performed with GraphPad Prism (Versions 8.4.3 and 10.2.3, GraphPad Software, San Diego, USA). Data are presented as mean ± standard deviation (SD) unless stated otherwise. For comparisons of more than two groups with a single factor (e.g. quantification of single- vs. double-positive cytospin fractions in Fig. 1 C, basal glycolysis, compensatory glycolysis and ATP production in Fig. 6 ), a one-way ANOVA was applied followed by Tukey’s multiple-comparison test. For experiments with two independent factors (e.g. passage number and pathological background in colony formation rate, flow-cytometric marker expression across passages, viability and cytotoxicity assays, and Annexin-V staining in Figs. 2 – 5 ), a two-way ANOVA was used. When appropriate, Sidak’s or Tukey’s post-hoc tests were performed to determine pairwise differences. When datasets contained missing values or unequal sample sizes across repeated measures (e.g. HT-I-56 expression across passages in Supp. Figure 4 ), a mixed-effects model (restricted maximum likelihood) was employed to account for random effects of patient ID, followed by Tukey’s multiple-comparison test. Paired comparisons within the same donor (e.g. pre- vs. post-cryopreservation viability) were analysed using two-tailed paired Student’s t-tests. Significance was defined as p < 0.05. Exact p-values are reported in the figure legends with the following notation: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). All experiments included at least duplicate technical replicates, and no data were excluded. Results Magnetic bead sorting yields highly pure AT-II and AT-I epithelial cells From 62 patients (21 controls, 15 IPF, 26 secondary fibrosis), AT-II and AT-I epithelial cells were successfully isolated by MACS against cell-specific plasma membrane markers HT-II-280 or HT-I-56 (Fig. 1 A). Immunofluorescence of cytospins directly after magnetic sorting showed 89% HT-II-280 + and 91.3% proSP-C + cells with a total of 83.7% double-positive population in HT-II-280-sorted cells on average (Fig. 1 B–C). This underlines a significant contrast (****p < 0.0001) to unsorted lung cells (3.1% HT-II-280 + and 49.4% proSP-C + cells) displaying a 2.9% double-positive population in total lung cells. Furthermore, also flow cytometry confirmed strong enrichment of HT-II-280 + and proSP-C + cells compared to unsorted tissue (Fig. 1 D, Supp. Figure 1 ). In a similar sorting approach, similarly, the isolation of AT-I cells yielded 93.0% HT-I-56 + cells (Fig. 1 E–F) in contrast to 16.7% HT-I-56 + cells in the unsorted lung cell fraction (***p = 0.0079), indicating robust applicability of the MACS strategy for alveolar epithelial subsets. This isolation workflow was robust across > 60 patient samples and can be readily adopted for large-scale tissue cohorts, supporting reproducibility and transferability of the approach. HT-II-280 + cells form 3D alveolospheres with high colony formation efficiency and diverse morphology To optimize 3D culturing conditions for primary human HTII-280⁺ alveolar type II (AT-II) cells, we initially adopted the protocol by Konishi et al. employing a 1:1 ratio of SFFF medium and Matrigel. While this ratio supported initial dome formation, the larger dome volumes (100–130 µl) used in our setup gradually collapsed, causing organoids to sink and contact the rigid plastic surface. Upon contact, cells at the dome–plastic interface lost their cuboidal morphology, detached from the surrounding matrix, and began spreading along the surface. These structural changes were accompanied by the appearance of flattened, basaloid-like epithelial cells after 7–10 days of culture (Supplementary Fig. 2). Increasing the proportion of Matrigel to a 5:8 (medium:Matrigel) ratio stabilized dome geometry, prevented sinking, and maintained proper embedding of organoids throughout 14-day culture. Therefore, freshly isolated AT-II cells embedded in Matrigel in a 5:8 rapidly formed organoids within several days (Fig. 2 A). Of note, the cultivation of HT-II-280 + cells resulted in the formation of both alveolospheres and airway organoids, as previously described [ 27 , 29 ] . Organoids formation reproducibly occurred from both control and fibrotic samples over multiple passages (Fig. 2 B-C). The colony formation rate (CFR) was initially lower in controls (41.5%) compared to fibrotic explants (65.4% at P0), but rose significantly in subsequent passages, reaching 79.6% in controls in P1 (****p < 0.0001) and 91.4–94.3% in fibrotic organoids in P1 and P2 (Fig. 2 C, P1 and P2: ****p < 0.0001). Fibrotic tissue-derived AT-II cells seem to form alveolospheres more efficiently across passages (P0: ****p < 0.0001; P2: ***p = 0.0002; P3:*p = 0.0373) than cells form tumor-distant “healthy” control tissue. Notably, colony formation efficiency was consistent across independent donors and passages, underlining the scalability of the culture system for biobanking and comparative ILD studies. Interestingly, organoids displayed heterogeneous morphologies, even within same passages and patients. Primary alveolospheres vary both in size, ranging from 50µm to 600 µm, as well in structural morphology: Monocystic and polycystic alveolospheres with visible lumina were frequently observed, whereas other organoids developed dense, lumenless spheroid structures as indicated by microcomputed tomography (Fig. 2 D–E). H&E staining confirmed alveolosphere-like architecture with epithelial polarity and diverse lumen formation (Fig. 2 F). This phenotypic variability was consistent across donors and passages, suggesting inherent heterogeneity of human AT-II progenitor-derived cultures. Organoids preserve AT-II identity across passages with limited AT-I differentiation Despite being physiologically diverse, immunofluorescence of whole mount organoids and cryosections demonstrated robust expression of HT-II-280 and proSP-C up to passage 2 (Fig. 3 A–C). On a single cell level, flow cytometry revealed that 82% of these cells in organoids express HT-II-280 + in the plasma membrane at P0 on average (75.9%[control], 88.5% [fibrotic]). Of note, HT-II-280 + expression declined to 65.6% at P3 on average in patient cohort (*p = 0.0423; Fig. 3 D-E, Supp. Figure 3 ). However, this reduction was more pronounced in controls (55.8%) than in fibrotic organoids (75.3%, *p = 0.0305). In stark contrast, proSP-C expression remained on a high level and consistently stable across passages and independent of tissue origin (79% [control] 89% [fibrotic] in P0; 84% [control] 93% [fibrotic] in P1; 83% [control] 88% [fibrotic] in P2; 83% [control] 80% [fibrotic] in P3) (Fig. 3 F–G). Hence, we can confirm that the system reliably maintains AT-II identity and can be reproduced across multiple laboratories. A moderate increase in AT-I marker HT-I-56 was detected by P3 (3.7% at P0 vs. 15.1% at P3, p = 0.0316; Supp. Figure 4 ). Dual-positive cells (proSP-C + /HT-I-56 + ) appeared in later passages, suggesting limited AT-II–AT-I transition while maintaining overall AT-II dominance. Lamellar bodies confirm AT-II ultrastructural identity LysoTracker® staining consistently identified lamellar body (LB)-positive cells by histology and flow cytometry across passages, with 89.7% LB + at P0 and 80.5% at P3 independent of tissue origin(Fig. 4 A–C). TEM further provided ultrastructural confirmation of AT-II identity, revealing numerous LBs, nuclei, mitochondria, and apical microvilli (Fig. 4 D–H). Dense-core LB granules were abundant in both control- and fibrosis-derived organoids. LB-like material was also observed in some organoid lumina, reflecting surfactant-related activity. The presence of lamellar bodies across all patient-derived cultures provides a reproducible ultrastructural hallmark of AT-II fidelity, further validating this model as a reliable epithelial platform. Cryopreservation preserves viability and organoid-forming capacity Following cryostorage at − 150°C for ≥ 28 days, revitalized und re-domed AT-II cells retained high viability and colony-forming potential. Annexin-V staining showed a small increase in apoptotic cells for control-derived organoids (*p = 0.0044) but not for fibrotic samples (p = 0.6497; Fig. 5 A–B). LDH-assays indicate that cytotoxicity remained unchanged after thawing (Fig. 5 C). Moreover, WST-1 assays confirmed preserved metabolic activity (Fig. 5 D). Concordantly, Live/Dead staining of alveolospheres formed after revitalization showed minimal cell death based on cell surface evaluation, similar to fresh cultures (Fig. 5 E). Together, these findings demonstrate the suitability of cryopreserved AT-II organoids for biobanking. Fibrotic AT-II organoids undergo glycolytic reprogramming The increased colony formation efficiency observed in fibrotic AT-II organoids suggested enhanced proliferative activity, which often coincides with metabolic shifts toward glycolysis. In idiopathic pulmonary fibrosis (IPF), alveolar epithelial cells exhibit profound mitochondrial dysfunction and glycolytic reprogramming, which are thought to sustain epithelial activation and maladaptive repair responses [ 35 – 36 ] . Moreover, glycolysis and lactate signaling have been implicated in driving fibrogenic processes under hypoxic and stress conditions [ 37 ] , while enhanced glycolytic activity was recently shown to be essential for energy production and regenerative capacity in alveolar stem cells [ 38 ] . Given these observations and the reported decline in regenerative potential of AT-II cells with disease progression [ 39 ] , we sought to determine whether our organoid model recapitulates these metabolic alterations by analyzing glycolytic and oxidative metabolism using extracellular flux assays. Indeed, extracellular flux analysis revealed enhanced glycolysis in fibrotic organoids compared to controls. In Detail, extracellular Acidification Rate (ECAR) was consistently higher in IPF- and secondary fibrosis-derived alveolospheres (Fig. 6 A–B). Interestingly, glycolysis was significantly (3-fold) elevated in AT-II organoids derived from IPF tissue vs healthy control tissue (*p = 0.0201) after glucose injection while same tendency was present in alveolospheres cultured from tissue of secondary fibrosis cases (1.4-fold increase; p = 0.7577). Upon oligomycin stimulation, IPF organoids displayed 3.5-fold higher compensatory glycolysis (*p = 0.0109), while secondary fibrosis organoids showed a 1.6-fold increase (p = 0.6455) in glycolytic capacity relative to controls (Fig. 6 C). OCR remained largely unchanged, suggesting preferential reliance on glycolysis over oxidative phosphorylation. ATP rate assays further demonstrated that fibrotic organoids generated significantly more ATP, with IPF samples producing 2.2-fold more (*p = 0.0477) and secondary fibrosis samples 1.7-fold more (p = 0.1228) ATP than controls (Fig. 6 D–E). Notably, the additional ATP was predominantly glycolytic, contributing 77.9% of total ATP in IPF, 63.3% in secondary fibrosis, and 42.9% in control AT-II organoids. Patient-to-patient heterogeneity was evident, as shown in Supplementary Figs. 5–6. Discussion In this study, we established a robust, cell-line-free, patient-derived 3D lung organoid model generated from primary human alveolar type II (AT-II) epithelial cells isolated from fibrotic and non-fibrotic lung tissues. Using HT-II-280-based magnetic bead sorting, adapted from Konishi et al. [ 33 ] , we achieved high-purity AT-II cell populations that were expanded in serum- and feeder-free conditions to form alveolospheres. These organoids recapitulated key AT-II features—proSP-C production, HT-II-280 localization, lamellar body formation—and preserved cell identity across passages. Compared to existing in vitro systems relying on iPSCs, immortalized lines, or murine cells [ 26 , 28 , 40 ] , our approach provides lineage fidelity and patient-specific disease signatures. A major strength of our model is the high cellular purity and phenotypic stability. More than 80% of cells within alveolospheres remained HT-II-280 + or proSP-C + across 3D-culture passaging, consistent with human AT-II organoid data [ 30 ] . Although HT-II-280–based magnetic sorting provided a highly enriched AT-II population prior to culturing, purity was not absolute. Immunofluorescence of cytospins revealed ~ 89% HT-II-280⁺ cells, while flow cytometry directly following sorting also detected strong enrichment of HT-II-280⁺ cells, however indicating that a small fraction of non–AT-II cells remained after isolation. These observations are consistent with the moderate colony formation rate (CFR) observed in the initial passage (P0), whereas both CFR and AT-II marker-specific labeling in flow cytometry notably increased in P1, suggesting that clonal expansion under serum- and feeder-free (SFFF) conditions—supplemented with proliferative boosters and AT-I inhibitors—further selects for bona fide AT-II progenitors during early culture. Ultrastructural analysis of organoids revealed abundant lamellar bodies, confirming mature AT-II identity [ 16 , 17 ] . Thus, the combination of magnetic enrichment and medium-driven selection in adapted matrigel-culturing likely achieves a higher functional purity than sorting alone. With our chosen ratio of matrigel to SFFF for initital dome pouring, we in addition overcome mechanical influences on epithelial fate: Matrix stiffness and mechanical cues are key regulators of alveolar epithelial differentiation in fibrotic lung tissue. It has been shown that increased rigidity of the extracellular matrix can induce AT-II cell dedifferentiation and emergence of aberrant basaloid states, as also observed in vivo in fibrotic regions of IPF [ 6 , 8 ] . Our in vitro findings mirror this behavior, as direct contact of organoids with the rigid plastic surface triggered loss of AT-II morphology and basaloid-like transformation. Increasing the Matrigel content to a 5:8 ratio prevented this effect by maintaining a compliant, fully embedded 3D environment. This adjustment thus represents a simple but essential refinement that preserves epithelial identity and enhances reproducibility in long-term culture of primary human AT-II organoids. Furthermore, differentiation into AT-I occurred solely modestly in later passages, with emergence of HT-I-56+/proSP-C + intermediates, indicating retained progenitor potential as described in other alveolar regeneration models [ 41 – 42 ] . Importantly, these results were reproducible across a large number of patient samples, supporting the transferability of this platform as a reliable technical resource. We also observed morphological heterogeneity of alveolospheres, which could be categorized as lumen-containing (type A) or dense lobular (type B), similar to murine AT-II-derived spheroids [ 18 ] and alveolar epithelial progenitor-derived structures [ 14 ] . Despite using highly enriched HT-II-280⁺ ATII cells, we occasionally observed airway-like organoids in addition to alveolospheres. This may reflect inclusion of epithelial progenitors with dual potential [ 18 ] , the influence of tissue origin (distal versus bronchiolar regions), and culture conditions that permit airway cell outgrowth, e.g. the addition of FGF10, consistent with previous reports on human airway organoids [ 29 ] .and HT-II-280⁺-derived cultures [ 27 ] . Rather than a limitation, such morphological heterogeneity reflects the target tissue and patient-specific biology and increases the translational relevance of the model for personalized medicine approaches. An important advance is the demonstration that organoids retained structural integrity and viability after cryopreservation. Cryostorage and successful revitalization were reproducible across both fibrotic and control tissues, demonstrating suitability for standardized biobanking and inter-center sharing. Following storage at − 150°C, we observed no loss of organoid-forming efficiency, viability, or metabolic activity, supporting the feasibility of biobanking. To our knowledge, this is among the first reports of successful cryorecovery of primary human AT-II organoids while preserving epithelial identity and functionality. This reproducibility across both control and fibrotic samples underscores the scalability of the approach and highlights its potential for inter-center biobanking, harmonized disease modeling, and standardized drug testing pipelines. Most notably, we identified a pronounced consistent metabolic shift in fibrotic AT-II organoids—especially those from IPF—towards aerobic glycolysis, establishing the organoids as a reproducible human platform to capture disease-specific energetic reprogramming. While extracellular flux assays have been used in murine epithelial organoids [ 43 ] , their application to primary human AT-II organoids from ILD patients is novel. By adapting extracellular flux analysis protocols from intestinal organoid studies [ 34 ] , we showed that IPF-derived alveolospheres exhibited elevated basal ECAR, enhanced compensatory glycolysis, and increased glycolytic ATP production. This recapitulates the “Warburg-like” metabolic phenotype described in IPF epithelial cells, driven by upregulated LDHA and PDK1, lactate accumulation, and reduced mitochondrial pyruvate dehydrogenase activity [ 24 , 35 – 36 ] . Because this phenotype was consistently detected across independent donors, our study provides a reproducible framework to interrogate metabolic reprogramming in patient-derived lung epithelium. The uncoupling of glycolysis from oxidative phosphorylation suggests disease-associated mitochondrial dysfunction and adaptation to high energetic demands of proliferation and ECM remodeling [ 26 , 37 ] . Sun et al. [ 25 ] reported that PDK1-driven lactate accumulation suppresses mitochondrial pyruvate dehydrogenase, reinforcing glycolytic flux. Metabolic studies in IPF have also shown decreased late glycolytic intermediates and elevated lactate [ 37 ] . Such metabolic rewiring is implicated in epithelial-to-mesenchymal transition (EMT), apoptosis resistance, and profibrotic signaling. The PDK1–HIF-1α axis promotes glycolysis-dependent myofibroblast differentiation under hypoxic/inflammatory stress [ 38 ] . Recent organoid-based work by Choi et al . [ 39 ] further demonstrated decreased regenerative capacity of IPF-derived AT-II cells with disease progression, highlighting the clinical significance of metabolic dysfunction. As a novel cellular model, some limitations must be noted. First, the model relies on end-stage explants, limiting insights into early pathogenic events [ 5 ] . Second, patient heterogeneity, despite normalization, introduces variability in sorting efficiency and expansion during cultivation. Third, while canonical markers validated AT-II identity, transcriptomic profiling is needed to capture sublineage states [ 41 , 44 ] . Incorporating stromal or immune components via co-culture or organoid-on-chip approaches could improve physiological relevance and enable modeling of epithelial–mesenchymal and epithelial–immune interactions [ 31 , 45 – 46 ] . Furthermore, possible transdifferentiation of AT-II cells into KRT5 + basal cells under profibrotic mesenchymal or TGF-β signals [ 32 ] underscores the need to explore epithelial plasticity in IPF organoids. By allowing direct scalable and reproducible comparison of fibrotic versus control AT-II organoids under patient-matched conditions, our model offers a platform to interrogate epithelial dysfunction and therapeutic responses for mechanistic studies, multicenter validation, and translational drug screening. Conclusion Together, our findings establish a reproducible, feeder-free 3D organoid model of primary human AT-II cells that preserves lineage identity and captures fibrotic metabolic reprogramming. These organoids retained ultrastructural identity and remained viable after cryopreservation, enabling long-term storage and recovery. Importantly, fibrotic organoids consistently displayed a glycolytic shift, reflecting disease-specific metabolic reprogramming. By combining reproducibility, scalability, and compatibility with cryopreservation, this model provides a transferable resource for biobanking, multicenter validation, and translational drug discovery. Beyond serving as a disease model, our platform establishes a technical foundation for future precision medicine approaches in interstitial lung diseases. Abbreviations ABCA3 – ATP-binding cassette sub-family A member 3 ANOVA – Analysis of variance AT-I – Alveolar type I cell AT-II– Alveolar type II cell ATP – Adenosine triphosphate CFR – Colony formation rate DAPI – 4′,6-diamidino-2-phenylindole DMSO – Dimethyl sulfoxide DZL – Deutsches Zentrum für Lungenforschung (German Center for Lung Research) ECAR – Extracellular acidification rate ECM – Extracellular matrix EGF – Epidermal growth factor EMT – Epithelial–mesenchymal transition ERS – European Respiratory Society FACS – Fluorescence-activated cell sorting FGF-10 – Fibroblast growth factor 10 GT – Goat antibody H&E – Hematoxylin and eosin HIF-1α – Hypoxia-inducible factor 1-alpha Hopx – Homeodomain-only protein x HT-I-56 – Human alveolar type I cell marker 56 HT-II-280 / HT-II-280 – Human alveolar type II cell marker 280 IF – Immunofluorescence ILD – Interstitial lung disease IPF – Idiopathic pulmonary fibrosis iPSC – Induced pluripotent stem cell KRT5 – Keratin 5 LB – Lamellar body LDH – Lactate dehydrogenase LDHA – Lactate dehydrogenase A LAMP3 – Lysosomal-associated membrane protein 3 MACS – Magnetic-activated cell sorting MHH – Medizinische Hochschule Hannover MS – Mouse antibody (context: anti-HT-II-280 ms monoclonal MT – Mitochondria MV – Microvilli NC – Nucleus NKX2-1 / TTF-1 – NK2 homeobox 1 / Thyroid transcription factor-1 OCR – Oxygen consumption rate P – Passage PDK1 – Pyruvate dehydrogenase kinase 1 PKM2 – Pyruvate kinase isoform M2 proSP-C – Pro-surfactant protein C RB – Rabbit antibody (context: anti-proSP-C rb polyclonal) RotAA – Rotenone & antimycin A SFTPB – Surfactant protein B SFTPC / SP-C – Surfactant protein C TEM – Transmission electron microscopy TGF-β – Transforming growth factor beta TTF-1 – Thyroid transcription factor-1 (NKX2-1) WST-1 – Water-soluble tetrazolium-1 Declarations Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies involving human participants were reviewed and approved by the Ethic Committee of MHH (ethic votes 8867_BO_K_2020 and 10194_BO_K_2022) and followed the rules of the Declaration of Helsinki of 1975. All patients provided written informed consent to participate in this study. Author contributions Idea and conceptualization: LJS, JR, SSCH, JCK, LN Funding acquisition: JCK, LN, MMH Clinical diagnosis validation and human tissue acquisition: CW, MK, RE, CPM, FI, PZ Methodology: LJS, JR, SMJ, MK, RE, CPM Data acquisition: LJS, JR, LK Data analysis and interpretation: LJS, JR Writing—original draft: LJS Writing—review: All authors contributed to the article and approved the submitted version. Funding This study was funded by the German Center for Lung Research (Deutsches Zentrum für Lungenforschung, DZL) and the Else-Kröner-Fresenius Foundation. Competing interest The authors declare no conflicts of interest. References Lederer DJ, Martinez FJ. 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Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1PatientCohort.xlsx Table. 1. Patient cohort. Demographics, diagnosis and experimental utilization of material for this study. Supplementarymethods.docx SupplementaryFiguresATIIPaper.pdf Supp. Fig. 1. Gating Strategy of HT-II-280 + cells following MACS Flow cytometry gating strategy for freshly MACSed primary AT-II cells in Fig. 1D. Each Isotype control and stainings were gated to exclude debris (Lane A), focus on single cells (Lane B) and then gate on FITC + cells (Lane C). Exemplary display of unsorted fractions, positive fractions labelled for HT-II-280 and positive fractions labelled for proSP-C. Supp. Fig. 2 Titration of medium to matrigel ration for 3d dome culturing The appropriate ratio for 14d 3D-culturing of freshly isolated HT-II-280+ cells was determined by matrigel titration experiments. When implementing culturing for human primary AT-II cells from fibrotic explants, we sarted by utilizing 1:1 ratio of matrigel and AT-II cell culture medium SFFF as previously published by Konishi et al. However, considering domes with larger volume 100-130µl and corresponding larger surface area, the 1:1 ratio led to sinking of organoids inside the dome and direct contact to plastic well, resulting in the transformation of HT-II-280+ cells to aberrant basaloid cells after 7-10 days of culturing (right panel). Upon initial contact with plastic, visible flaking of AT-II cells occurs on the surface of the existing organoids (pink arrows). After prolonged sinking and contact with rigid plastic, visible spreading and transformation into aberrant basoids occurs (white arrowheads). Therefore, ratio was adjusted to 5:8 for 3D culturing for this study, resulting in proper embedding of cells for prolonged cultivation (left panel). Supp. Fig. 3. Gating Strategy of HT-II-280 + cell 3D organoid culture Flow cytometry gating strategy for AT-II cells cultured as organoids in matrigel domes corresponding to Fig. 3D and 3F. Gating strategy included exclusion of debris (Lane A), focus on single cells (Lane B) and gating on FITC+ cells in isotype control in sample A (Lane C) or staining in sample B (Lane D) per measurement. Similar gating was applied for flow cytometry of Lysotracker in Fig. 4B, Annexin V staining in Fig.5A and HT-I-56 staining in Supp. Fig. 3. Supp. Fig. 4. Minor differentiation of AT-II cells to AT-I cells during 3D-culturing in later passages A. Whole mount immunofluorescence staining displays certain HT-I-56 positive cells on the surface of AT-II alveolospheres (example from fibrotic explant, passage 2). Representative examples of N=5 organoid cultures from IPF patients and N=4 tumor-distant tissues from resections. Though majority of surface level cells from the lung organoid display proSP-C-production in P2, single cells display protein expression of the AT-I cell-specific marker. Scale bar=50µm. B. Direct comparison of HT-I-56 expression utilizing immunolabeling of cryosections from the same tissue/patient (fibrotic explant) between P0 and P2 highlights certain differentiation of AT-II to AT-I cells in vitro in 3D culture. Data from N=3 IPF and N=1 healthy controls from tumor-distant tissues, P0-P2. Scale bar=50µm. C. Total cells from lung organoids were separated into single cell suspension and labeled with HT-I-56 ms IgG1 and gt anti-mg IgG1 (γ chain) Alexa Fluor-488 (displayed in FITC channel) for flow cytometry. Example from the same tissue/patient (fibrotic explant) between P0 and P2. Scale bar=50µm. D. Quantification of HT-I-56 expression. Statistical analysis showed significant difference in HT-I-56 expression between P0 and P3 in cells derived from fibrotic explants (Mixed effects analysis F=3.834 *, p=0.0387; Tukey‘s multiple comparison test *=0.0319), but no difference between cells derived from fibrotic explants (IPF and secondary fibrosis, turquoise, N=4-5 or healthy tumor-distant lung tissues (red, N=3). Supp. Fig. 5. Entire cohort of extracellular flux measurements: Glycolysis stress test Cohort for Seahorse assays displayed heterogeneity which causes high deviation between patients per group in Fig. 5B and 5C. For illustration, ECAR data, OCR data and corresponding basal glycolysis and compensatory glycolysis is shown for all 18 patients (N=7 controls “GLR”, N=6 secondary fibrosis “GLE” [sec. Fib] and N=5 IPF “GLE” [IPF]). For more information on tissue background, see Table 1. Supp. Fig. 6. Entire cohort of extracellular flux measurements: ATP Rate Assay Cohort for Seahorse assays displayed heterogeneity which causes high deviation between patients per group in Fig. 5E. For illustration, ECAR data, OCR data, and corresponding ATP rates, divided in glycoATP and mitoATP, are shown for all 18 patients (N=7 controls “GLR”, N=6 secondary fibrosis “GLE” [sec. Fib], and N=5 IPF “GLE” [IPF]). For more information on tissue background, see Supplementary Table 1. Supp.Table1SFFFMedia.xlsx Supp. Table.1. Serum-free feeder-free medium. Components for generation of AT-II cell culturing medium. Cite Share Download PDF Status: Published Journal Publication published 07 Mar, 2026 Read the published version in Respiratory Research → Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 24 Nov, 2025 Reviews received at journal 04 Nov, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers invited by journal 23 Oct, 2025 Editor assigned by journal 23 Oct, 2025 Submission checks completed at journal 23 Oct, 2025 First submitted to journal 21 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7913465","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538750140,"identity":"7054d5b7-32e9-41d6-b7c5-e3bea6d7ab6e","order_by":0,"name":"Lara-Jasmin Schröder","email":"data:image/png;base64,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","orcid":"","institution":"Hannover Medical School","correspondingAuthor":true,"prefix":"","firstName":"Lara-Jasmin","middleName":"","lastName":"Schröder","suffix":""},{"id":538750142,"identity":"ef599922-f082-4065-bd6b-557ddecde264","order_by":1,"name":"Julia Rückoldt","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Rückoldt","suffix":""},{"id":538750144,"identity":"1cdcd433-40f6-4d67-a8c9-ec8c31df971f","order_by":2,"name":"Stephanie Schubert","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Schubert","suffix":""},{"id":538750147,"identity":"51c02297-bf4f-4f8c-91f0-381110255bb5","order_by":3,"name":"Lars Knudsen","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Lars","middleName":"","lastName":"Knudsen","suffix":""},{"id":538750148,"identity":"0e77c239-c416-4744-a836-9c4caaa812d3","order_by":4,"name":"Sabina-Marija Janciauskiene","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Sabina-Marija","middleName":"","lastName":"Janciauskiene","suffix":""},{"id":538750150,"identity":"d7d9e177-9408-48dd-b9c5-9d3764fbafe7","order_by":5,"name":"Christopher Werlein","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Werlein","suffix":""},{"id":538750151,"identity":"9e5c5209-c552-46c5-85b8-01ce83d04873","order_by":6,"name":"Mareike Knoll","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Mareike","middleName":"","lastName":"Knoll","suffix":""},{"id":538750152,"identity":"7d4e5dae-bbc1-49de-845c-13235aac9190","order_by":7,"name":"Regina Engelhardt","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Regina","middleName":"","lastName":"Engelhardt","suffix":""},{"id":538750153,"identity":"e43a1e20-5ba9-494e-910d-21d30680e758","order_by":8,"name":"Christina Petzold-Mügge","email":"","orcid":"","institution":"Hannover Medical School","correspondingAuthor":false,"prefix":"","firstName":"Christina","middleName":"","lastName":"Petzold-Mügge","suffix":""},{"id":538750154,"identity":"15f2b1c3-8484-4363-ab27-ce724bb1658b","order_by":9,"name":"Marius M. 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1","display":"","copyAsset":false,"role":"figure","size":1105401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation of AT-II and AT-I epithelial cells by magnetic bead sorting.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eSchematics of sorting-based isolation. Cohort of this study included 62 lung tissues derived from N=15 IPF and N=26 secondary pulmonary fibrosis patients in addition to N=21 tumor-distant, pathologically inconspicuous control tissues. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eExemplary display of cytospins\u003cstrong\u003e \u003c/strong\u003eof 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=20µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e. Quantification of HT-II-280\u003csup\u003e+\u003c/sup\u003e proSP-C\u003csup\u003e+\u003c/sup\u003e cells/total to DAPI\u003csup\u003e+\u003c/sup\u003e 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\u003csup\u003e+\u003c/sup\u003e vs. HT-II-280\u003csup\u003e+\u003c/sup\u003e proSP-C\u003csup\u003e+\u003c/sup\u003e cells; ns, p=0.0631 for proSP-C\u003csup\u003e+\u003c/sup\u003e vs. HT-II-280\u003csup\u003e+\u003c/sup\u003e proSP-C\u003csup\u003e+\u003c/sup\u003e cells). However, HT-II-280\u003csup\u003e+ \u003c/sup\u003eare strongly enriched after sorting (****, p\u0026lt;0.0001) alongside proSP-C\u003csup\u003e+\u003c/sup\u003e 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eExemplary flow cytometry measurement before and directly after MACS-based sorting illustrates strong enrichment of HT-II-280\u003csup\u003e+ \u003c/sup\u003ecells 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 Figure 1. Representative data of total N=7 (N=3 healthy tissue donors and N=4 fibrotic explants).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e 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=20µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eQuantification 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\u003csup\u003e+ \u003c/sup\u003efollowing sorting (***, p=0.00452). Representative picture of 5 tissues (N=2 from healthy tumor-distant control tissue, N=3 from fibrotic explants).\u003c/p\u003e","description":"","filename":"Figures21.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/c2a0bb88dccdd5917812bcb3.png"},{"id":95120112,"identity":"1f036b35-9087-4110-bb7f-354ba57091af","added_by":"auto","created_at":"2025-11-04 13:53:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1844508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology and growth pattern of primary human AT-II organoids during 3D culturing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eRepresentative bright-field images of an alveolosphere (upper panel) and airway organoid (lower panel) emerging from HT-II-280\u003csup\u003e+\u003c/sup\u003e cells. Formation of AT-II alveolospheres vs. airway organoids in 3D matrigel domes cultures over the course of 14 days in vitro following HT-II-280\u003csup\u003e+\u003c/sup\u003ecell population isolation. Scale bars in all images equals 50 µm except last overview of airway organoid (=100 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eClonal expansion of organoid culture at 12 d in P1. Bright-field images, Scale bar=250µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eColony formation rate (CFR) of AT-II cells from initial passage 0 to passage 3. For quantification, for each patient ID, 5 bright field images from B. were counted at 40x magnification for the relative number of colonies (called alveolospheres) on day 7 of culture. Data points represent mean of N=18 fibrotic explant and N=12 tumor distant control tissue-derived AT-II organoid cultures per passage. Red= healthy background, blue= fibrotic background. Following two-way ANOVA (****, p\u0026lt;0.0001), Tukey’s multiple comparisons were performed to illustrate the difference in colony formation between fibrotic and healthy tissue-derived AT-II cells each passage (P0: ****, p\u0026lt;0.0001, P1: n; p=0.3197,P2: ***p=0.0002, P3: *p=0.0373) as indicated (black asterisks), while blue (fibrotic, ****, p\u0026lt;0.0001) and red asterisks (control, ****, p\u0026lt;0.0001) show significant increase of formed colonies between P0 und P1 for both backgrounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eExemplary plastic overview of lung organoids in microcomputed tomography derived from a fibrotic explant at 12 days post-isolation (first passage). NanoTom scan images. Representative examples of N=2 organoid cultures from IPF patients and N=2 tumor-distant tissues from resections. Zoom-in windows highlight heterogeneous sizing and variance in shape of organoids per passage. Scale bar=400µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eVolume rendering of lung organoids during microcomputed tomography revealed the presence of smaller (50-70 µm diameter) mono- (yellow arrowheads) and polycystic (green arrowheads) alveolospheres with lumen. Other smaller (\u0026lt;100µm, purple arrows) and larger organoids (\u0026gt;150µm, pink arrows) are completely filled/layered out with AT-II cells and display a honeycomb-structure. Representative examples of N=2 organoid cultures from IPF patients and N=2 tumor-distant tissues from resections. Scale bar=100µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eHistopathology of alveolar organoid cultures. Representative image of lung organoid FFPE sections in hematoxylin and eosin (H\u0026amp;E) staining. Organoids from N=7 fibrotic patients and N=3 healthy tissues were investigated. Scale bar= 100 µm. Mono- (yellow arrowheads) and polycystic (green arrowheads) organoids with lumen. Smaller (\u0026lt;100 µm, purple arrows) and larger organoids (\u0026gt;100 µm, pink arrows) with lumenless-structure.\u003c/p\u003e","description":"","filename":"Figures22.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/bf3cb74a6072dcf63c4cfd50.png"},{"id":95120060,"identity":"8d754d51-e02b-48c9-aeb7-db92a55e3875","added_by":"auto","created_at":"2025-11-04 13:53:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1843208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrimary human lung organoids from explants and tumor-distant control tissues display expression of AT-II specific markers after 3D-culturing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eWhole mount immunofluorescence labeling illustrates that the surface of lung organoids from AT-II cells respond to HT-II-280 and proSP-C expression (example from P1, 12d, fibrotic explant). All replicates (n=5 fibrotic n=3 healthy controls from tumor-distant tissues, P0-P2) express proSP-C and HT-II-280 at the surface. Scale bar=100 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eRepresentative images from cryosection immunolabeling of alveolospheres indicate expression of secreted proSP-C throughout the lumen of lung organoids (and) and in the extracellular space. Representative images of proSP-C staining on larger (top panel), and smaller alveolospheres (middle and bottom panel) with diverse morphology. N=7 fibrotic N=4 healthy controls from tumor-distant tissues (P0-P2). Scale bar=50 µm or 20 µm, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eFluorescent images demonstrate the predominant localization of HT-II-280 in the apical plasma membrane of ATII cells (red) as well as some intracytoplasmic staining and luminal accumulation / apical shedding. Representative images of staining on larger polycystic (top panel), small monocystic (middle panel) or small polycystic (bottom panel) alveolospheres from N=6 fibrotic and N=6 healthy controls from tumor-distant tissues (P0-P2). Scale bar=50 µm or 20 µm, respectively\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eRepresentative flow cytometry measurement for HT-II-280 on human AT-II cells from organoids tracked from initial passage 0 up to passage 3. Primary anti-HT-II-280 ms IgM was conjugated to gt anti-ms IgM Alexa Fluor-488 (displayed in FITC channel). Full gating strategy for P0 is displayed in Supplementary Figure 2. Example from cells dissociated from alveolospheres at Passage 0, fibrotic explant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Quantification of HT-II-280 expression in cells from lung organoids measured by flow cytometry until passage 3. Statistical analysis showed significant difference in HT-II-280 expression between P0 and P3 (two-way ANOVA *, p=0.0426; Tukey‘s multiple comparison test *), but no difference between cells derived from fibrotic explants (IPF and secondary fibrosis, turquoise) or healthy tumor-distant lung tissues (red) except in P3 (two-way ANOVA *, p=0.0288; Tukey‘s multiple comparison test *). Cells derived from N=5-6 fibrotic tissues and N=5-6 tumor-distant control tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eRepresentative flow cytometry measurement for proSP-C in human AT-II cells from organoids displayed per passage. Primary anti- proSP-C rb polyclonal was conjugated to gt anti-rb (H+L) Alexa Fluor-488 (displayed in FITC channel). Full gating strategy for P0 is displayed in Supplementary Figure 2. Example from cells dissociated from alveolospheres at Passage 0, fibrotic explant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Quantification of proSP-C expression in total living cells from lung organoids in same flow cytometry measurement over the course of 3 passages. Statistical analysis showed neither significant difference in proSP-C expression between P0 and P3 (two-way ANOVA ns, p=0.4915) nor difference between cells derived from fibrotic explants (IPF and secondary fibrosis, turquoise) or healthy tumor-distant lung tissues (red). Cells derived from N=5-7 fibrotic tissues and N=4-6 tumor-distant control tissues.\u003c/p\u003e","description":"","filename":"Figures23.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/8a4d4beab5d7533769bb8955.png"},{"id":95120059,"identity":"ddde3c52-8460-4e80-b657-67d2d05fc1a8","added_by":"auto","created_at":"2025-11-04 13:53:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1830463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence of lamellar bodies in primary human lung organoids indicates purity of AT-II cells in alveolospheres.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eWhole-mount live immunofluorescence staining exhibits the presence of acidic lysosome-related organelles by Lysotracker in cells of the surface layer of AT-II organoids (example from P1, 12d, fibrotic explant). All replicates (N=5 fibrotic tissues, N=4 healthy controls from tumor-distant tissues, P0-P3) of organoid cultures show signals for intracellular Lysotracker dye in live cell imaging of alveolospheres (upper panel). Lower panel shows differences between highly-positive Lysotracker signals in alveolospheres (pink arrows) vs. less pronounced presence of lamellar bodies in airway organoids (grey arrows). Data shown are representative for three independent experiments. Scale bar=100 µm or 250 µm, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eRepresentative flow cytometry gates for Lysotracker Green DND-26 staining of cell derived from alveolospheres at P2, 10 d from a fibrotic lung explant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eQuantification of Lysotracker-positive cell population over different culturing passages. Two-way ANOVA analysis did not demonstrate significant alterations of presumably lamellar body-containing AT-II cells between passages P0 and P3 (ns, p=0.1391). Cells derived from N=5-6 fibrotic tissues and N=4-6 tumor-distant control tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eRepresentative transmission electron microscopy (TEM) images of monocystic alveolosphere (small); Overview: scale bar= 10μm; higher magnifications as indicated. NC= nucleus. Microvilli (MVs, red window and red arrow heads) were observed in spheres at the apical lumen. Mitochondria (MT) displayed with blue arrow heads. A monocystic organoid displays accumulations of numerous lamellar bodies on ultrastructural level (LB, black). Black window shows intracellular LBs, while yellow and green window display extracellular LBs. Qualitative analysis displays LBs in all investigated samples (N=4 fibrotic tissues, N=2 healthy controls from tumor-distant tissues, P0-P1). High magnification images in TEM show single AT-II cell from a monocystic organoid. NC (white), MV (red arrow heads), MT (blue arrow heads) and LB (black). Scale bar= 5μm. \u003cstrong\u003eE. \u003c/strong\u003eDetails of a single AT-II cells within a monocystic alveolosphere. Scale bar= 5μm Presence of NC, MTs, LBs and MVs. Structural diversity between AT-II alveolospheres displayed in TEM is further displayed by small lumenless \u003cstrong\u003e(F.), \u003c/strong\u003esmall polycystic \u003cstrong\u003e(G.) \u003c/strong\u003eor large honeycomb-structured alveolospheres containing intermediate filaments \u003cstrong\u003e(H.); \u003c/strong\u003escale bars each 10μm.\u003c/p\u003e","description":"","filename":"Figures24.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/05bfb5c445b6b6a4d4e6adaa.png"},{"id":95120074,"identity":"ecbc7c53-30ea-4332-bc7f-f4f877eeca1e","added_by":"auto","created_at":"2025-11-04 13:53:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":671394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRevitalization and re-culturing of cryopreserved AT-II cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Exemplary FACS analysis of dead (Annexin V\u003csup\u003e+\u003c/sup\u003e) AT-II cells derived from alveolospheres prior and post cryopreservation for 28d at -150 °C. Freshly isolated and cultured AT-II cells from P0 (at 4d culturing) vs re-cultured cells 4 d following revitalization, P1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Quantification of Annexin V\u003csup\u003e+\u003c/sup\u003e AT-II cells from FACS analysis. Patient-matched (N=5 fibrotic tissues and N=5 tumor-distant control tissues) comparisons of dead cell percentage before and after cryopreservation (each 4d in P0 and P1) showed difference as displayed by two-way ANOVA (F(1,8)=3.9284, *p=0.0044 for effect of pathological background, F(1,8)=2.567, p=0.1478 for effect of cryopreservation and F(1,8)=20.88, p**=0.0018 for combined effects) and Sidak‘s multiple comparison test (indicated asterisks).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Quantification of cytotoxicity measured by LDH-Assay in cell culture media of matrigel domes containing AT-II cells. Release of LDH activity was measured at 4 d in P0 (freshly cultured) and 4 d in P1 (post cryopreservation). LDH values are represented as the relative amount of released LDH activity compared with the totally lysed control. N=6 fibrotic tissues and N=6 tumor-distant control tissues. Data are expressed as means ± SD. Two-way ANOVA (F(1,10)=0.04342, p=0.8391 for effect of pathological background, F(1,10)=0.5485, p=0.4770 for effect of cryopreservation and F(1,10)=0.4050, p=0.5388 for combined effects) and Sidak‘s multiple comparison test. Release relative to positive control which is generated by incubating one matrigel dome for 45 min with SFFF medium containing 1% Triton X-100.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Quantification of viability before and after cryopreservation by WST-1 assay’s absorbance at 450-690 nm. Measurements at 4 d in P0 (freshly cultured) and 4 d in P1 (post cryopreservation). N=6 fibrotic tissues and N=6 tumor-distant control tissues. Two-way ANOVA (F(1,10)=0.2.441, P=0.1493 for effect of pathological background, F(1,10)=1.249, p=0.2899 for effect of cryopreservation and F(1,10)=0.0021, p=0.9642 for combined effects) and Sidak‘s multiple comparison test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Representative images of immunofluorescent Live/Dead staining of AT-II organoid surface. Alveolospheres were re-cultured for 12 d following revitalization (P1. Patient-matched alveolospheres from initial culture P0 serve as reference. In total, N=4 fibrotic and N=4 healthy tissue-derived AT-II cells. Nuclei were stained with DAPI.\u003c/p\u003e","description":"","filename":"Figures25.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/4241c7c037e6807e589bdeca.png"},{"id":95120107,"identity":"ab2621f4-2831-44bd-80b9-9a0bf9d0c9cb","added_by":"auto","created_at":"2025-11-04 13:53:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":84625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFibrotic AT-II cells within primary organoids increase glycolysis and decrease level of mitochondrial respiration during end-stage disease.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Exemplary measurement of extracellular acidification rate (ECAR) (A) and corresponding O\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;consumption rate (OCR) was measured for primary ATII cells cultured for 12 d. IPF, secondary fibrosis or healthy origin. Data including 18 patients (N= 7 tumor-distant control tissues, N= 6 sec. fibrosis and N= 5 IPF tissues) highlights trend towards increased glycolytic activity of IPF-derived AT-II organoids in basal glycolysis rates (\u003cstrong\u003eB.\u003c/strong\u003e) and significant difference in compensatory glycolysis rates (\u003cstrong\u003eC.\u003c/strong\u003e). Data is normalized to control. Basal glycolysis (\u003cstrong\u003eB.\u003c/strong\u003e) assessed by one-way ANOVA (F=4.954;*p=0.0233) followed by Tukey’s multiple comparison test (*p=0.0201 for control vs. IPF; p=0.7577 for control vs. sec. Fibrosis; p=0.0852 for sec. fibrosis vs. IPF). Compensatory glycolysis (\u003cstrong\u003eC.\u003c/strong\u003e) assessed by one-way ANOVA (F=5.905; *p=0.0128) followed by Tukey’s multiple comparison test (*, p=0.0109 for control vs. IPF; p=0.6455 for control vs. sec. fibrosis; p=0.0679 for sec. fibrosis vs. IPF). To assess the ATP production rate, measurements were conducted utilizing oligomycin addition to the assay media followed by RotAA addition. Representative ECAR und OCR data (\u003cstrong\u003eD.\u003c/strong\u003e) was utilized to calculate the total ATP production rate (\u003cstrong\u003eE.\u003c/strong\u003e) based on total glyoATP and mitoATP production. The level of calculated proportions of total basal ATP production rates corresponding to glyoATP and mitoATP rate are shown in Fig. E based on same 18 patients. Assessment of ATP production by one-way ANOVA (F= 3.881; *p=0.401) followed by Tukey’s multiple comparison test (*p=0.0477 for control vs. IPF; p=0.1228 for control vs. sec. Fibrosis; p=0.4473 for sec. fibrosis vs. IPF).\u003c/p\u003e","description":"","filename":"Figures26.png","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/3d04fa34026f11320f317ab5.png"},{"id":104251987,"identity":"7e84b319-2857-4b81-a6e5-604d3c161874","added_by":"auto","created_at":"2026-03-09 16:16:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8900434,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/d3617e37-324c-4bbd-be8e-6a204ad68652.pdf"},{"id":95120087,"identity":"fd041494-4b45-4b2c-973e-3fac94353def","added_by":"auto","created_at":"2025-11-04 13:53:28","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable. 1. Patient cohort. \u003c/strong\u003eDemographics, diagnosis and experimental utilization of material for this study.\u003c/p\u003e","description":"","filename":"Table1PatientCohort.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/98cd3bef80d582d35079fada.xlsx"},{"id":95224810,"identity":"d13f0dbf-b97f-4c5a-bbd9-74bdbc4072df","added_by":"auto","created_at":"2025-11-05 16:24:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26088,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/a61fcbe2631d9db5656a1945.docx"},{"id":95120061,"identity":"eaf67bc5-d8a2-4211-90e5-0f23e74dc272","added_by":"auto","created_at":"2025-11-04 13:53:26","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1240095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupp. Fig. 1. Gating Strategy of HT-II-280\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells following MACS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry gating strategy for freshly MACSed primary AT-II cells in Fig. 1D. Each Isotype control and stainings were gated to exclude debris (Lane A), focus on single cells (Lane B) and then gate on FITC\u003csup\u003e+\u003c/sup\u003e cells (Lane C). Exemplary display of unsorted fractions, positive fractions labelled for HT-II-280 and positive fractions labelled for proSP-C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Supp. Fig. 2 Titration of medium to matrigel ration for 3d dome culturing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe appropriate ratio for 14d 3D-culturing of freshly isolated HT-II-280+ cells was determined by matrigel titration experiments. When implementing culturing for human primary AT-II cells from fibrotic explants, we sarted by utilizing 1:1 ratio of matrigel and AT-II cell culture medium SFFF as previously published by \u003cem\u003eKonishi et al.\u003c/em\u003e However, considering domes with larger volume 100-130µl and corresponding larger surface area, the 1:1 ratio led to sinking of organoids inside the dome and direct contact to plastic well, resulting in the transformation of HT-II-280+ cells to aberrant basaloid cells after 7-10 days of culturing (\u003cstrong\u003eright panel\u003c/strong\u003e). Upon initial contact with plastic, visible flaking of AT-II cells occurs on the surface of the existing organoids (pink arrows). After prolonged sinking and contact with rigid plastic, visible spreading and transformation into aberrant basoids occurs (white arrowheads). Therefore, ratio was adjusted to 5:8 for 3D culturing for this study, resulting in proper embedding of cells for prolonged cultivation \u003cstrong\u003e(left panel)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eSupp. Fig. 3. Gating Strategy of HT-II-280\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell 3D organoid culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry gating strategy for AT-II cells cultured as organoids in matrigel domes corresponding to Fig. 3D and 3F. Gating strategy included exclusion of debris (Lane A), focus on single cells (Lane B) and gating on FITC+ cells in isotype control in sample A (Lane C) or staining in sample B (Lane D) per measurement. Similar gating was applied for flow cytometry of Lysotracker in Fig. 4B, Annexin V staining in Fig.5A and HT-I-56 staining in Supp. Fig. 3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Supp. Fig. 4.\u003c/strong\u003e \u003cstrong\u003eMinor differentiation of AT-II cells to AT-I cells during 3D-culturing in later passages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Whole mount immunofluorescence staining displays certain HT-I-56 positive cells on the surface of AT-II alveolospheres (example from fibrotic explant, passage 2). Representative examples of N=5 organoid cultures from IPF patients and N=4 tumor-distant tissues from resections. Though majority of surface level cells from the lung organoid display proSP-C-production in P2, single cells display protein expression of the AT-I cell-specific marker. Scale bar=50µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Direct comparison of HT-I-56 expression utilizing immunolabeling of cryosections from the same tissue/patient (fibrotic explant) between P0 and P2 highlights certain differentiation of AT-II to AT-I cells in vitro in 3D culture. Data from N=3 IPF and N=1 healthy controls from tumor-distant tissues, P0-P2. Scale bar=50µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Total cells from lung organoids were separated into single cell suspension and labeled with HT-I-56 ms IgG1 and gt anti-mg IgG1 (γ chain) Alexa Fluor-488 (displayed in FITC channel) for flow cytometry. Example from the same tissue/patient (fibrotic explant) between P0 and P2. Scale bar=50µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e Quantification of HT-I-56 expression. Statistical analysis showed significant difference in HT-I-56 expression between P0 and P3 in cells derived from fibrotic explants (Mixed effects analysis F=3.834 *, p=0.0387; Tukey‘s multiple comparison test *=0.0319), but no difference between cells derived from fibrotic explants (IPF and secondary fibrosis, turquoise, N=4-5 or healthy tumor-distant lung tissues (red, N=3).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eSupp. Fig. 5. Entire cohort of extracellular flux measurements: Glycolysis stress test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCohort for Seahorse assays displayed heterogeneity which causes high deviation between patients per group in Fig. 5B and 5C. For illustration, ECAR data, OCR data and corresponding basal glycolysis and compensatory glycolysis is shown for all 18 patients (N=7 controls “GLR”, N=6 secondary fibrosis “GLE” [sec. Fib] and N=5 IPF “GLE” [IPF]). For more information on tissue background, see Table 1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eSupp. Fig. 6. Entire cohort of extracellular flux measurements: ATP Rate Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCohort for Seahorse assays displayed heterogeneity which causes high deviation between patients per group in Fig. 5E. For illustration, ECAR data, OCR data, and corresponding ATP rates, divided in glycoATP and mitoATP, are shown for all 18 patients (N=7 controls “GLR”, N=6 secondary fibrosis “GLE” [sec. Fib], and N=5 IPF “GLE” [IPF]). For more information on tissue background, see Supplementary Table 1.\u003c/p\u003e","description":"","filename":"SupplementaryFiguresATIIPaper.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/bce25d6f1983155ab9f93953.pdf"},{"id":95120102,"identity":"492184f8-461b-4225-8618-d9e9ff3a394c","added_by":"auto","created_at":"2025-11-04 13:53:28","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupp. Table.1. Serum-free feeder-free medium. \u003c/strong\u003eComponents for generation of AT-II cell culturing medium.\u003c/p\u003e","description":"","filename":"Supp.Table1SFFFMedia.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7913465/v1/1658e7d521f21c80676ca378.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eHuman Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease (ILD) characterized by excessive extracellular matrix (ECM) deposition, loss of alveolar structure, and respiratory failure. The disease primarily affects older adults and has a poor prognosis, with a median survival of only 3\u0026ndash;5 years after diagnosis \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Current anti-fibrotic therapies can slow progression but neither halt nor reverse fibrosis \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The lack of curative options highlights the urgent need for human-derived disease models that recapitulate IPF pathogenesis \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlveolar type II (AT-II) epithelial cells play a central role in alveolar homeostasis and fibrotic remodeling. These cuboidal surfactant-producing cells constitute\u0026thinsp;~\u0026thinsp;5% of the alveolar surface, secrete surfactant, regulate immune responses, and act as progenitors for alveolar type I (AT-I) cells \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Under normal conditions, AT-II cells self-renew and transdifferentiate into AT-I cells after injury \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. In IPF, however, AT-II cells undergo apoptosis, senescence, or aberrant activation, contributing to chronic epithelial damage and fibrosis \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. In addition to these processes, AT-II cells in fibrotic lungs can give rise to aberrant basaloid cells, a transitional epithelial state observed in distal fibrotic regions of IPF \u003csup\u003e[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Recent studies, including single-cell transcriptomic analyses, suggest that matrix stiffening and altered mechanical cues drive this basaloid differentiation program \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Such stiffness-induced epithelial transitions have been demonstrated in vitro and in vivo, where direct contact with rigid substrates promotes loss of AT-II identity and activation of basal markers \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. They may further sustain the fibrotic niche by secreting profibrotic cytokines, undergoing epithelial\u0026ndash;mesenchymal transition (EMT), and serving as progenitors of myofibroblasts \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. AT-II cells can be identified by lineage-defining markers such as surfactant proteins (SFTPC, SFTPB), ABCA3 \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, LAMP3 \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, NKX2-1/TTF-1, and the surface marker HT-II-280 used for human isolation \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Morphologically, the presence of lamellar bodies is a characteristic hallmark of AT-II cells \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. AT-I cells, in contrast, cover\u0026thinsp;~\u0026thinsp;95% of the alveolar surface and express RAGE/ AGER, HT-I-56, and HOPX\u003csup\u003e17\u0026ndash;18]\u003c/sup\u003e. In addition to AT-II\u0026ndash;derived fibroblast-like states, injured AT-I cells in IPF adopt dysfunctional/senescent programs that secrete pro-fibrotic mediators (e.g., TGF-β, CTGF, WNT ligands), thereby activating fibroblasts and amplifying ECM deposition; AT-I\u0026ndash;restricted regulators such as AGER (RAGE) and caveolin-1 further modulate this crosstalk, and their dysregulation has been linked to heightened fibrosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent studies showed that IPF-derived AT-II cells undergo metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis, a \u0026ldquo;Warburg-like\u0026rdquo; phenotype. This is linked to mitochondrial dysfunction, elevated glycolytic enzyme expression (LDHA, PDK1), lactate accumulation, and suppression of pyruvate dehydrogenase via the PDK1\u0026ndash;HIF-1α axis \u003csup\u003e[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Such energetic shifts are thought to drive fibrotic remodeling and represent potential therapeutic targets.\u003c/p\u003e\u003cp\u003eOrganoids\u0026mdash;self-organizing 3D epithelial culture systems\u0026mdash;have become valuable models for disease research. Pulmonary organoids derived from fetal, adult, or induced pluripotent stem cell (iPSC) sources can recapitulate alveolar architecture and function \u003csup\u003e[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. They allow long-term expansion, passaging, and cryopreservation while maintaining lineage identity, enabling applications in disease modeling, drug testing, and personalized medicine \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Several lung organoid models reproduced features of fibrosis, epithelial plasticity, and anti-fibrotic responses \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Compared to 2D cultures, organoids better preserve epithelial polarity, surfactant secretion, and progenitor functions. However, robust primary human AT-II organoid models from end-stage fibrotic lungs are still lacking. Most existing models rely on murine or iPSC-derived epithelium and do not reflect the native metabolic or regenerative state of diseased AT-II cells.\u003c/p\u003e\u003cp\u003eMoreover, current systems often lack reproducibility across laboratories and limited scalability, restricting their broader translational use. A reproducible, feeder-free human AT-II model that allows cryopreservation and biobanking would therefore be highly valuable for precision medicine and cross-center validation.\u003c/p\u003e\u003cp\u003eHere, we establish a defined feeder-free 3D culture system for primary human HT-II-280\u0026thinsp;+\u0026thinsp;AT-II cells isolated from both healthy and fibrotic (IPF and secondary fibrosis) lung tissue adapted from previous work by \u003cem\u003eKonishi et al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eKatsura et al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. We aimed to (i) maintain AT-II identity across passages using canonical markers (HT-II-280, proSP-C, lamellar bodies), (ii) assess cryopreservation and potential for organoid biobanking, and (iii) evaluate metabolic differences between healthy and fibrotic AT-II organoids using extracellular flux analysis. We hypothesized that fibrotic AT-II cells exhibit enhanced glycolytic metabolism compared to controls, reflecting disease-specific metabolic reprogramming. By providing a robust and transferable protocol, our study delivers a reproducible human lung organoid resource that supports mechanistic and translational research in ILD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient cohort and tissue collection\u003c/h2\u003e\u003cp\u003eFor this study, we analysed 62 lung tissues (22 females, 40 males; average age 60\u0026thinsp;\u0026plusmn;\u0026thinsp;12 years), including 41 fibrotic explants (15 IPF, 26 secondary pulmonary fibrosis) and 21 tumor-distant \u0026ldquo;healthy\u0026rdquo; controls. Tissues were used either for one or multiple investigations. Donors provided written informed consent, and the study was approved by the Hannover Medical School ethics committee (8867_BO_K_2020, 10194_BO_K_2022). Patient demographics and tissue utilization are summarized in Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation of primary alveolar epithelial cells\u003c/h3\u003e\n\u003cp\u003ePrimary human alveolar type II (AT-II) and type I (AT-I) cells were isolated from freshly explanted fibrotic or tumor-distant \u0026ldquo;healthy\u0026rdquo; control lung tissue by magnetic-activated cell sorting (MACS) using the AT-II marker HT-II-280 and the AT-I marker HT-I-56, respectively \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. All steps were performed on ice or at 4\u0026deg;C unless otherwise specified and all buffers and consumables were pre-cooled.\u003c/p\u003e\u003cp\u003ePeripheral lung tissue was dissected free of pleura, bronchi, and large vessels, mechanically minced, and enzymatically digested (Miltenyi Biotec, gentleMACS and No. 130-110-201 Multi Tissue Dissociation Kit 1) [Miltenyi Biotec, Cat. No. 130-110-201]). After sequential filtration (100/70 \u0026micro;m), centrifugation (450 g, 7 min, 4\u0026deg;C), and erythrocyte lysis with ACK lysis buffer (Thermo Fisher Scientific/Gibco, Cat. No. A10492-01) followed by sequential filtration (40 \u0026micro;m), cells were washed and resuspended in MACS buffer (1% BSA, 2 mM EDTA in PBS).\u003c/p\u003e\u003cp\u003eFollowing Fc receptor blockade, (10 \u0026micro;L/sample; Miltenyi Biotec) for 15 min at 4\u0026deg;C, cells were incubated with 5 \u0026micro;L mouse IgM anti-HT-II-280 antibody (Terrace Biotech, Cat. No. TB-27AHT2-280; 1:50 in MACS buffer) for 1 h at 4\u0026deg;C with gentle rotation (60 rpm). 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.\u003c/p\u003e\u003cp\u003eCells were washed with 1.5 mL MACS buffer, centrifuged, and resuspended in 2 mL MACS buffer containing anti-mouse IgM or anti-mouse IgG MicroBeads (Miltenyi Biotec; 20 \u0026micro;L beads per 10\u003csup\u003e7\u003c/sup\u003e cells, diluted 1:10 in MACS buffer). Bead incubation was carried out for 30 min at 4\u0026deg;C with gentle rotation. After washing, suspensions were applied to pre-equilibrated LS columns in an OctoMACS separator. Negative fractions were collected as flow-through, while positive fractions were eluted after column removal from the magnet. Both fractions were pelleted, resuspended in MACS buffer, and used for counting or downstream applications. MACS was selected over FACS to reduce shear stress and preserve viability for organoid culture.\u003c/p\u003e\u003cp\u003eBoth positive and negative fractions were pelleted (450 g, 7 min, 4\u0026deg;C) and resuspended in 200 \u0026micro;L MACS buffer for counting or immediate downstream applications.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3D organoid culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMACS-isolated HT-II-280\u003csup\u003e+\u003c/sup\u003e AT-II cells were pelleted (450 g, 7 min, 4\u0026deg;C) and resuspended in serum-free, feeder-free (SFFF) medium based on Advanced DMEM/F-12 (Gibco, Cat 12634028) supplemented with a variety of proliferation enhancers e.g. GSK3β-Inhibitor CHIR99021 and AT-I cell differentiation inhibitors such as ROCK-Inhibitor Y-27632, TGF-β-Receptor I (ALK5)-Inhibitor SB431542 and p38 MAPK-Inhibitor BIRB796 (see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). SFFF medium was adapted from \u003cem\u003eKonishi et al.\u003c/em\u003e \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e using the same components but with modified concentrations to optimize maintenance of AT-II cell phenotype and increased proliferation. Cells in SFFF medium were mixed with Corning\u0026reg; Matrigel\u0026reg; Growth Factor Reduced Basement Membrane Matrix (Cat. No. 356231) at a 5:8 ratio (cell suspension:matrigel) and seeded as 100\u0026ndash;130 \u0026micro;L domes in 24-well plates (60,000 cells/dome) in initial passage (P0). After polymerization (20\u0026ndash;30 min, 37\u0026deg;C), 500 \u0026micro;L SFFF medium was added per well. For the first 4\u0026ndash;5 days, IL-1β was added to enhance alveolosphere formation. Culture medium also contained EGF and FGF-10 to support Wnt and ERK/AKT pathway activation, essential for AT-II expansion. Organoids were grown for up to 16 days and passaged up to P3, with medium exchanged every 2\u0026ndash;3 days.\u003c/p\u003e\u003cp\u003eTo generate single-cell suspensions for passaging of AT-II cells, organoids were segregated using TrypLE\u0026trade; Select Enzyme (Gibco, Cat. No. 12563011). Per matrigel dome, 500 \u0026micro;L TrypLE\u0026trade; Select Enzyme were utilized to firstly mechanically disrupt domes and incubate them at 37\u0026deg;C for 15\u0026ndash;30 min with regard to organoid sizes and numbers. Suspensions were again mechanically disrupted, washed 3x in PBS, and centrifuged at 500 g for 5 min before further processing.\u003c/p\u003e\u003cp\u003e\u003cem\u003eTechnical note\u003c/em\u003e: For all histological investigations and especially for flow cytometry labeling, we strongly recommend the utilization of Cell Recovery Solution (Corning, Cat. No.: 354253) before further processing. 300 \u0026micro;L Cell Recovery Solution were added to the domes to dissolve organoids from matrigel for more surface efficient stainings (e.g. such as for HT-II-280 labeling) and minimization on unlabeled cells in FACS to to residual matrigel. Organoids free of matrigel can further undergo immediate trypsination.\u003c/p\u003e\n\u003ch3\u003eCryopreservation\u003c/h3\u003e\n\u003cp\u003eFor cryopreservation, organoids were mechanically and enzymatically dissociated as described above. Up to 500,000 single cells were resuspended in 1 ml cryomedium (70% FBS, 20% SFFF medium, 10% DMSO, added dropwise). Cryomedium was incubated with AT-II for 20min at RT to allow for proper diffusion of DMSO. Cryovials were placed in a Mr. Frosty\u0026trade; container at \u0026minus;\u0026thinsp;80\u0026deg;C and transferred after 24 h to \u0026minus;\u0026thinsp;150\u0026deg;C for long-term storage. To assess safety and viability, samples were stored\u0026thinsp;\u0026ge;\u0026thinsp;28 days at \u0026minus;\u0026thinsp;150\u0026deg;C. For recovery, cells were thawed in a 37\u0026deg;C water bath for 2 min, diluted in 14 ml Advanced DMEM/F-12 at room temperature, incubated for 10 min, centrifuged (7 min, 450 g), and directly reseeded in Matrigel at a 5:8 ratio.\u003c/p\u003e\n\u003ch3\u003eIn-depth characterization of alveolar organoids\u003c/h3\u003e\n\u003cp\u003eTo confirm the identity and purity of alveolar organoids over multiple passages and to evaluate cryostorage potential, a multitude of techniques was applied including conventional histology, microcomputed tomography, transmission electron microscopy, image analysis, immunofluorescence \u0026amp; flow cytometry as well as viability assays. For the sake of space, these techniques are described in detail in the supplement.\u003c/p\u003e\n\u003ch3\u003eExtracellular flux assays\u003c/h3\u003e\n\u003cp\u003eTo assess the metabolic properties of fibrotic vs control organoids, glycolytic capacity and ATP production of fibrotic and healthy tissue-derived AT-II cells measured by extracellular flux assays using the \u003cem\u003eAgilent Seahorse XF HS Mini Analyzer S7852A\u003c/em\u003e platform (Agilent, Santa Clara, CA). Organoids from P0-P1 were separated to single-cell suspension using trypsination as previously described in \u003cem\u003emethod section\u003c/em\u003e. Next, 12,000 cells from P1-P2 were plated 10\u0026ndash;12 d prior to experiments in each well of \u003cem\u003eXFp PDL Miniplates\u003c/em\u003e (Agilent, Cat. No.: 03722\u0026thinsp;\u0026minus;\u0026thinsp;100) in matrigel domes with 50 \u0026micro;l total volume as suggested by \u003cem\u003eLudikhuize et al\u003c/em\u003e. \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e and provided with SFFF media. By day of experiment, each well therefore roughly contains 85,000-120,000 cells as organoids. Samples were prepared in duplets. Cartridges were prepared corresponding to the manufacturer\u0026rsquo;s instructions for \u003cem\u003eSeahorse XFp Glycolysis Stress Test Kit\u003c/em\u003e (Agilent, Cat. No.: 103017-100) and \u003cem\u003eSeahorse XFp Real-Time ATP Rate Assay Kit\u003c/em\u003e (Agilent, Cat. No.: 103591-100). On the day of experiment, assay media were freshly prepared. SFFF was aspirated from matrigel domes exhibiting normal sized alveolospheres after cultivation miniplates and exchanged with 100 \u0026micro;l assay media 45 min prior to measurement, resulting in a total volume of 150 \u0026micro;l per well. Of note, after serial titration experiments for \u003cem\u003eSeahorse XFp Glycolysis Stress Test Kit\u003c/em\u003e, we increased the concentrations of glucose to 15 mM, oligomycin to 5 \u0026micro;M and 2-DG to 200 mM per well, each resuspended in assay medium. Each injection port contained 20 \u0026micro;l volume. We measured 3 cycles each basal curve, glycolysis, and glycolytic capacity followed by 5 cycles of glycolytic reserve asessement. Similarly, for \u003cem\u003eSeahorse XFp Real-Time ATP Rate Assay Kit\u003c/em\u003e, oligomycin (5\u0026micro;M) and Retenone\u0026thinsp;+\u0026thinsp;Antimycin A (2\u0026micro;M) per well were utilized in higher concentration than recommended from Agilent for measurement of 2D cell culture and with a volume of 20 \u0026micro;l per injection port. These concentrations are roughly in line with concentration utilized to measure extracellular flux of intestinal organoids by \u003cem\u003eLudikhuize et al\u003c/em\u003e. \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. ATP production were assessed by 3 cycles each basal ECAR and OCR, inhibition of mitochondrial ATP synthesis, and, finally, mitochondrial-associated acidification. Following measurements, organoids were collected from matrigel using Cell Recovery Solution (Corning, Cat. No.: 354253) and harvested for DNA isolation using QIAamp DNA micro kit (Qiagen, Cat. No.:56304) and samples were measured by Nanodrop to enable a normalization of experimental values relative to approximated number of cells. Additionally, BrdU assays (Cell Signaling Technology, Cat. No.: 6813S) were performed to confirm unaltered cell proliferation and viability post-extracellular flux assays (data not shown).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed with GraphPad Prism (Versions 8.4.3 and 10.2.3, GraphPad Software, San Diego, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) unless stated otherwise. For comparisons of more than two groups with a single factor (e.g. quantification of single- vs. double-positive cytospin fractions in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, basal glycolysis, compensatory glycolysis and ATP production in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e), a one-way ANOVA was applied followed by Tukey\u0026rsquo;s multiple-comparison test. For experiments with two independent factors (e.g. passage number and pathological background in colony formation rate, flow-cytometric marker expression across passages, viability and cytotoxicity assays, and Annexin-V staining in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e), a two-way ANOVA was used. When appropriate, Sidak\u0026rsquo;s or Tukey\u0026rsquo;s post-hoc tests were performed to determine pairwise differences. When datasets contained missing values or unequal sample sizes across repeated measures (e.g. HT-I-56 expression across passages in Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e), a mixed-effects model (restricted maximum likelihood) was employed to account for random effects of patient ID, followed by Tukey\u0026rsquo;s multiple-comparison test. Paired comparisons within the same donor (e.g. pre- vs. post-cryopreservation viability) were analysed using two-tailed paired Student\u0026rsquo;s t-tests. Significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Exact p-values are reported in the figure legends with the following notation: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (****). All experiments included at least duplicate technical replicates, and no data were excluded.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eMagnetic bead sorting yields highly pure AT-II and AT-I epithelial cells\u003c/h2\u003e\u003cp\u003eFrom 62 patients (21 controls, 15 IPF, 26 secondary fibrosis), AT-II and AT-I epithelial cells were successfully isolated by MACS against cell-specific plasma membrane markers HT-II-280 or HT-I-56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunofluorescence of cytospins directly after magnetic sorting showed 89% HT-II-280\u003csup\u003e+\u003c/sup\u003e and 91.3% proSP-C\u003csup\u003e+\u003c/sup\u003e cells with a total of 83.7% double-positive population in HT-II-280-sorted cells on average (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;C). This underlines a significant contrast (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) to unsorted lung cells (3.1% HT-II-280\u003csup\u003e+\u003c/sup\u003e and 49.4% proSP-C\u003csup\u003e+\u003c/sup\u003e cells) displaying a 2.9% double-positive population in total lung cells. Furthermore, also flow cytometry confirmed strong enrichment of HT-II-280\u003csup\u003e+\u003c/sup\u003e and proSP-C\u003csup\u003e+\u003c/sup\u003e cells compared to unsorted tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In a similar sorting approach, similarly, the isolation of AT-I cells yielded 93.0% HT-I-56\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;F) in contrast to 16.7% HT-I-56\u003csup\u003e+\u003c/sup\u003e cells in the unsorted lung cell fraction (***p\u0026thinsp;=\u0026thinsp;0.0079), indicating robust applicability of the MACS strategy for alveolar epithelial subsets. This isolation workflow was robust across \u0026gt;\u0026thinsp;60 patient samples and can be readily adopted for large-scale tissue cohorts, supporting reproducibility and transferability of the approach.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHT-II-280\u003csup\u003e+\u003c/sup\u003e cells form 3D alveolospheres with high colony formation efficiency and diverse morphology\u003c/h2\u003e\u003cp\u003eTo optimize 3D culturing conditions for primary human HTII-280⁺ alveolar type II (AT-II) cells, we initially adopted the protocol by \u003cem\u003eKonishi et al.\u003c/em\u003e employing a 1:1 ratio of SFFF medium and Matrigel. While this ratio supported initial dome formation, the larger dome volumes (100\u0026ndash;130 \u0026micro;l) used in our setup gradually collapsed, causing organoids to sink and contact the rigid plastic surface. Upon contact, cells at the dome\u0026ndash;plastic interface lost their cuboidal morphology, detached from the surrounding matrix, and began spreading along the surface. These structural changes were accompanied by the appearance of flattened, basaloid-like epithelial cells after 7\u0026ndash;10 days of culture (Supplementary Fig.\u0026nbsp;2). Increasing the proportion of Matrigel to a 5:8 (medium:Matrigel) ratio stabilized dome geometry, prevented sinking, and maintained proper embedding of organoids throughout 14-day culture.\u003c/p\u003e\u003cp\u003eTherefore, freshly isolated AT-II cells embedded in Matrigel in a 5:8 rapidly formed organoids within several days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Of note, the cultivation of HT-II-280\u003csup\u003e+\u003c/sup\u003e cells resulted in the formation of both alveolospheres and airway organoids, as previously described \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Organoids formation reproducibly occurred from both control and fibrotic samples over multiple passages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). The colony formation rate (CFR) was initially lower in controls (41.5%) compared to fibrotic explants (65.4% at P0), but rose significantly in subsequent passages, reaching 79.6% in controls in P1 (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and 91.4\u0026ndash;94.3% in fibrotic organoids in P1 and P2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P1 and P2: ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Fibrotic tissue-derived AT-II cells seem to form alveolospheres more efficiently across passages (P0: ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; P2: ***p\u0026thinsp;=\u0026thinsp;0.0002; P3:*p\u0026thinsp;=\u0026thinsp;0.0373) than cells form tumor-distant \u0026ldquo;healthy\u0026rdquo; control tissue. Notably, colony formation efficiency was consistent across independent donors and passages, underlining the scalability of the culture system for biobanking and comparative ILD studies.\u003c/p\u003e\u003cp\u003eInterestingly, organoids displayed heterogeneous morphologies, even within same passages and patients. Primary alveolospheres vary both in size, ranging from 50\u0026micro;m to 600 \u0026micro;m, as well in structural morphology: Monocystic and polycystic alveolospheres with visible lumina were frequently observed, whereas other organoids developed dense, lumenless spheroid structures as indicated by microcomputed tomography (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E). H\u0026amp;E staining confirmed alveolosphere-like architecture with epithelial polarity and diverse lumen formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This phenotypic variability was consistent across donors and passages, suggesting inherent heterogeneity of human AT-II progenitor-derived cultures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eOrganoids preserve AT-II identity across passages with limited AT-I differentiation\u003c/h2\u003e\u003cp\u003eDespite being physiologically diverse, immunofluorescence of whole mount organoids and cryosections demonstrated robust expression of HT-II-280 and proSP-C up to passage 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;C). On a single cell level, flow cytometry revealed that 82% of these cells in organoids express HT-II-280\u003csup\u003e+\u003c/sup\u003e in the plasma membrane at P0 on average (75.9%[control], 88.5% [fibrotic]). Of note, HT-II-280\u003csup\u003e+\u003c/sup\u003e expression declined to 65.6% at P3 on average in patient cohort (*p\u0026thinsp;=\u0026thinsp;0.0423; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E, Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, this reduction was more pronounced in controls (55.8%) than in fibrotic organoids (75.3%, *p\u0026thinsp;=\u0026thinsp;0.0305). In stark contrast, proSP-C expression remained on a high level and consistently stable across passages and independent of tissue origin (79% [control] 89% [fibrotic] in P0; 84% [control] 93% [fibrotic] in P1; 83% [control] 88% [fibrotic] in P2; 83% [control] 80% [fibrotic] in P3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u0026ndash;G). Hence, we can confirm that the system reliably maintains AT-II identity and can be reproduced across multiple laboratories.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA moderate increase in AT-I marker HT-I-56 was detected by P3 (3.7% at P0 vs. 15.1% at P3, p\u0026thinsp;=\u0026thinsp;0.0316; Supp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Dual-positive cells (proSP-C\u003csup\u003e+\u003c/sup\u003e/HT-I-56\u003csup\u003e+\u003c/sup\u003e) appeared in later passages, suggesting limited AT-II\u0026ndash;AT-I transition while maintaining overall AT-II dominance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eLamellar bodies confirm AT-II ultrastructural identity\u003c/h2\u003e\u003cp\u003eLysoTracker\u0026reg; staining consistently identified lamellar body (LB)-positive cells by histology and flow cytometry across passages, with 89.7% LB\u003csup\u003e+\u003c/sup\u003e at P0 and 80.5% at P3 independent of tissue origin(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;C). TEM further provided ultrastructural confirmation of AT-II identity, revealing numerous LBs, nuclei, mitochondria, and apical microvilli (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;H). Dense-core LB granules were abundant in both control- and fibrosis-derived organoids. LB-like material was also observed in some organoid lumina, reflecting surfactant-related activity. The presence of lamellar bodies across all patient-derived cultures provides a reproducible ultrastructural hallmark of AT-II fidelity, further validating this model as a reliable epithelial platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCryopreservation preserves viability and organoid-forming capacity\u003c/h2\u003e\u003cp\u003eFollowing cryostorage at \u0026minus;\u0026thinsp;150\u0026deg;C for \u0026ge;\u0026thinsp;28 days, revitalized und re-domed AT-II cells retained high viability and colony-forming potential. Annexin-V staining showed a small increase in apoptotic cells for control-derived organoids (*p\u0026thinsp;=\u0026thinsp;0.0044) but not for fibrotic samples (p\u0026thinsp;=\u0026thinsp;0.6497; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B). LDH-assays indicate that cytotoxicity remained unchanged after thawing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, WST-1 assays confirmed preserved metabolic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Concordantly, Live/Dead staining of alveolospheres formed after revitalization showed minimal cell death based on cell surface evaluation, similar to fresh cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Together, these findings demonstrate the suitability of cryopreserved AT-II organoids for biobanking.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eFibrotic AT-II organoids undergo glycolytic reprogramming\u003c/h2\u003e\u003cp\u003eThe increased colony formation efficiency observed in fibrotic AT-II organoids suggested enhanced proliferative activity, which often coincides with metabolic shifts toward glycolysis. In idiopathic pulmonary fibrosis (IPF), alveolar epithelial cells exhibit profound mitochondrial dysfunction and glycolytic reprogramming, which are thought to sustain epithelial activation and maladaptive repair responses \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Moreover, glycolysis and lactate signaling have been implicated in driving fibrogenic processes under hypoxic and stress conditions \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, while enhanced glycolytic activity was recently shown to be essential for energy production and regenerative capacity in alveolar stem cells \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Given these observations and the reported decline in regenerative potential of AT-II cells with disease progression \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, we sought to determine whether our organoid model recapitulates these metabolic alterations by analyzing glycolytic and oxidative metabolism using extracellular flux assays.\u003c/p\u003e\u003cp\u003eIndeed, extracellular flux analysis revealed enhanced glycolysis in fibrotic organoids compared to controls. In Detail, extracellular Acidification Rate (ECAR) was consistently higher in IPF- and secondary fibrosis-derived alveolospheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;B). Interestingly, glycolysis was significantly (3-fold) elevated in AT-II organoids derived from IPF tissue vs healthy control tissue (*p\u0026thinsp;=\u0026thinsp;0.0201) after glucose injection while same tendency was present in alveolospheres cultured from tissue of secondary fibrosis cases (1.4-fold increase; p\u0026thinsp;=\u0026thinsp;0.7577). Upon oligomycin stimulation, IPF organoids displayed 3.5-fold higher compensatory glycolysis (*p\u0026thinsp;=\u0026thinsp;0.0109), while secondary fibrosis organoids showed a 1.6-fold increase (p\u0026thinsp;=\u0026thinsp;0.6455) in glycolytic capacity relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). OCR remained largely unchanged, suggesting preferential reliance on glycolysis over oxidative phosphorylation.\u003c/p\u003e\u003cp\u003eATP rate assays further demonstrated that fibrotic organoids generated significantly more ATP, with IPF samples producing 2.2-fold more (*p\u0026thinsp;=\u0026thinsp;0.0477) and secondary fibrosis samples 1.7-fold more (p\u0026thinsp;=\u0026thinsp;0.1228) ATP than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;E). Notably, the additional ATP was predominantly glycolytic, contributing 77.9% of total ATP in IPF, 63.3% in secondary fibrosis, and 42.9% in control AT-II organoids. Patient-to-patient heterogeneity was evident, as shown in Supplementary Figs.\u0026nbsp;5\u0026ndash;6.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established a robust, cell-line-free, patient-derived 3D lung organoid model generated from primary human alveolar type II (AT-II) epithelial cells isolated from fibrotic and non-fibrotic lung tissues. Using HT-II-280-based magnetic bead sorting, adapted from \u003cem\u003eKonishi et al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, we achieved high-purity AT-II cell populations that were expanded in serum- and feeder-free conditions to form alveolospheres. These organoids recapitulated key AT-II features\u0026mdash;proSP-C production, HT-II-280 localization, lamellar body formation\u0026mdash;and preserved cell identity across passages. Compared to existing \u003cem\u003ein vitro\u003c/em\u003e systems relying on iPSCs, immortalized lines, or murine cells \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, our approach provides lineage fidelity and patient-specific disease signatures.\u003c/p\u003e\u003cp\u003eA major strength of our model is the high cellular purity and phenotypic stability. More than 80% of cells within alveolospheres remained HT-II-280\u003csup\u003e+\u003c/sup\u003e or proSP-C\u003csup\u003e+\u003c/sup\u003e across 3D-culture passaging, consistent with human AT-II organoid data \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Although HT-II-280\u0026ndash;based magnetic sorting provided a highly enriched AT-II population prior to culturing, purity was not absolute. Immunofluorescence of cytospins revealed\u0026thinsp;~\u0026thinsp;89% HT-II-280⁺ cells, while flow cytometry directly following sorting also detected strong enrichment of HT-II-280⁺ cells, however indicating that a small fraction of non\u0026ndash;AT-II cells remained after isolation. These observations are consistent with the moderate colony formation rate (CFR) observed in the initial passage (P0), whereas both CFR and AT-II marker-specific labeling in flow cytometry notably increased in P1, suggesting that clonal expansion under serum- and feeder-free (SFFF) conditions\u0026mdash;supplemented with proliferative boosters and AT-I inhibitors\u0026mdash;further selects for bona fide AT-II progenitors during early culture. Ultrastructural analysis of organoids revealed abundant lamellar bodies, confirming mature AT-II identity \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Thus, the combination of magnetic enrichment and medium-driven selection in adapted matrigel-culturing likely achieves a higher functional purity than sorting alone. With our chosen ratio of matrigel to SFFF for initital dome pouring, we in addition overcome mechanical influences on epithelial fate: Matrix stiffness and mechanical cues are key regulators of alveolar epithelial differentiation in fibrotic lung tissue. It has been shown that increased rigidity of the extracellular matrix can induce AT-II cell dedifferentiation and emergence of aberrant basaloid states, as also observed in vivo in fibrotic regions of IPF \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Our \u003cem\u003ein vitro\u003c/em\u003e findings mirror this behavior, as direct contact of organoids with the rigid plastic surface triggered loss of AT-II morphology and basaloid-like transformation. Increasing the Matrigel content to a 5:8 ratio prevented this effect by maintaining a compliant, fully embedded 3D environment. This adjustment thus represents a simple but essential refinement that preserves epithelial identity and enhances reproducibility in long-term culture of primary human AT-II organoids. Furthermore, differentiation into AT-I occurred solely modestly in later passages, with emergence of HT-I-56+/proSP-C\u0026thinsp;+\u0026thinsp;intermediates, indicating retained progenitor potential as described in other alveolar regeneration models \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Importantly, these results were reproducible across a large number of patient samples, supporting the transferability of this platform as a reliable technical resource.\u003c/p\u003e\u003cp\u003eWe also observed morphological heterogeneity of alveolospheres, which could be categorized as lumen-containing (type A) or dense lobular (type B), similar to murine AT-II-derived spheroids \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and alveolar epithelial progenitor-derived structures \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Despite using highly enriched HT-II-280⁺ ATII cells, we occasionally observed airway-like organoids in addition to alveolospheres. This may reflect inclusion of epithelial progenitors with dual potential \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, the influence of tissue origin (distal versus bronchiolar regions), and culture conditions that permit airway cell outgrowth, e.g. the addition of FGF10, consistent with previous reports on human airway organoids \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.and HT-II-280⁺-derived cultures \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Rather than a limitation, such morphological heterogeneity reflects the target tissue and patient-specific biology and increases the translational relevance of the model for personalized medicine approaches.\u003c/p\u003e\u003cp\u003eAn important advance is the demonstration that organoids retained structural integrity and viability after cryopreservation. Cryostorage and successful revitalization were reproducible across both fibrotic and control tissues, demonstrating suitability for standardized biobanking and inter-center sharing. Following storage at \u0026minus;\u0026thinsp;150\u0026deg;C, we observed no loss of organoid-forming efficiency, viability, or metabolic activity, supporting the feasibility of biobanking. To our knowledge, this is among the first reports of successful cryorecovery of primary human AT-II organoids while preserving epithelial identity and functionality. This reproducibility across both control and fibrotic samples underscores the scalability of the approach and highlights its potential for inter-center biobanking, harmonized disease modeling, and standardized drug testing pipelines.\u003c/p\u003e\u003cp\u003eMost notably, we identified a pronounced consistent metabolic shift in fibrotic AT-II organoids\u0026mdash;especially those from IPF\u0026mdash;towards aerobic glycolysis, establishing the organoids as a reproducible human platform to capture disease-specific energetic reprogramming. While extracellular flux assays have been used in murine epithelial organoids \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e, their application to primary human AT-II organoids from ILD patients is novel. By adapting extracellular flux analysis protocols from intestinal organoid studies \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, we showed that IPF-derived alveolospheres exhibited elevated basal ECAR, enhanced compensatory glycolysis, and increased glycolytic ATP production. This recapitulates the \u0026ldquo;Warburg-like\u0026rdquo; metabolic phenotype described in IPF epithelial cells, driven by upregulated LDHA and PDK1, lactate accumulation, and reduced mitochondrial pyruvate dehydrogenase activity \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Because this phenotype was consistently detected across independent donors, our study provides a reproducible framework to interrogate metabolic reprogramming in patient-derived lung epithelium.\u003c/p\u003e\u003cp\u003eThe uncoupling of glycolysis from oxidative phosphorylation suggests disease-associated mitochondrial dysfunction and adaptation to high energetic demands of proliferation and ECM remodeling \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eSun et al.\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e reported that PDK1-driven lactate accumulation suppresses mitochondrial pyruvate dehydrogenase, reinforcing glycolytic flux. Metabolic studies in IPF have also shown decreased late glycolytic intermediates and elevated lactate \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Such metabolic rewiring is implicated in epithelial-to-mesenchymal transition (EMT), apoptosis resistance, and profibrotic signaling. The PDK1\u0026ndash;HIF-1α axis promotes glycolysis-dependent myofibroblast differentiation under hypoxic/inflammatory stress \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Recent organoid-based work by \u003cem\u003eChoi et al\u003c/em\u003e. \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e further demonstrated decreased regenerative capacity of IPF-derived AT-II cells with disease progression, highlighting the clinical significance of metabolic dysfunction.\u003c/p\u003e\u003cp\u003eAs a novel cellular model, some limitations must be noted. First, the model relies on end-stage explants, limiting insights into early pathogenic events \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Second, patient heterogeneity, despite normalization, introduces variability in sorting efficiency and expansion during cultivation. Third, while canonical markers validated AT-II identity, transcriptomic profiling is needed to capture sublineage states \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Incorporating stromal or immune components via co-culture or organoid-on-chip approaches could improve physiological relevance and enable modeling of epithelial\u0026ndash;mesenchymal and epithelial\u0026ndash;immune interactions \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Furthermore, possible transdifferentiation of AT-II cells into KRT5\u003csup\u003e+\u003c/sup\u003e basal cells under profibrotic mesenchymal or TGF-β signals \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e underscores the need to explore epithelial plasticity in IPF organoids.\u003c/p\u003e\u003cp\u003eBy allowing direct scalable and reproducible comparison of fibrotic versus control AT-II organoids under patient-matched conditions, our model offers a platform to interrogate epithelial dysfunction and therapeutic responses for mechanistic studies, multicenter validation, and translational drug screening.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTogether, our findings establish a reproducible, feeder-free 3D organoid model of primary human AT-II cells that preserves lineage identity and captures fibrotic metabolic reprogramming. These organoids retained ultrastructural identity and remained viable after cryopreservation, enabling long-term storage and recovery. Importantly, fibrotic organoids consistently displayed a glycolytic shift, reflecting disease-specific metabolic reprogramming. By combining reproducibility, scalability, and compatibility with cryopreservation, this model provides a transferable resource for biobanking, multicenter validation, and translational drug discovery. Beyond serving as a disease model, our platform establishes a technical foundation for future precision medicine approaches in interstitial lung diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\" style=\"margin-right: calc(-1%); width: 101%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 59.6943%;\"\u003e\n \u003cp\u003eABCA3 \u0026ndash; ATP-binding cassette sub-family A member 3\u003c/p\u003e\n \u003cp\u003eANOVA \u0026ndash; Analysis of variance\u003c/p\u003e\n \u003cp\u003eAT-I \u0026ndash; Alveolar type I cell\u003c/p\u003e\n \u003cp\u003eAT-II\u0026ndash; Alveolar type II cell\u003c/p\u003e\n \u003cp\u003eATP \u0026ndash; Adenosine triphosphate\u003c/p\u003e\n \u003cp\u003eCFR \u0026ndash; Colony formation rate\u003c/p\u003e\n \u003cp\u003eDAPI \u0026ndash; 4\u0026prime;,6-diamidino-2-phenylindole\u003c/p\u003e\n \u003cp\u003eDMSO \u0026ndash; Dimethyl sulfoxide\u003c/p\u003e\n \u003cp\u003eDZL \u0026ndash; Deutsches Zentrum f\u0026uuml;r Lungenforschung (German Center for Lung Research)\u003c/p\u003e\n \u003cp\u003eECAR \u0026ndash; Extracellular acidification rate\u003c/p\u003e\n \u003cp\u003eECM \u0026ndash; Extracellular matrix\u003c/p\u003e\n \u003cp\u003eEGF \u0026ndash; Epidermal growth factor\u003c/p\u003e\n \u003cp\u003eEMT \u0026ndash; Epithelial\u0026ndash;mesenchymal transition\u003c/p\u003e\n \u003cp\u003eERS \u0026ndash; European Respiratory Society\u003c/p\u003e\n \u003cp\u003eFACS \u0026ndash; Fluorescence-activated cell sorting\u003c/p\u003e\n \u003cp\u003eFGF-10 \u0026ndash; Fibroblast growth factor 10\u003c/p\u003e\n \u003cp\u003eGT \u0026ndash; Goat antibody\u003c/p\u003e\n \u003cp\u003eH\u0026amp;E \u0026ndash; Hematoxylin and eosin\u003c/p\u003e\n \u003cp\u003eHIF-1\u0026alpha; \u0026ndash; Hypoxia-inducible factor 1-alpha\u003c/p\u003e\n \u003cp\u003eHopx \u0026ndash; Homeodomain-only protein x\u003c/p\u003e\n \u003cp\u003eHT-I-56 \u0026ndash; Human alveolar type I cell marker 56\u003c/p\u003e\n \u003cp\u003eHT-II-280 / HT-II-280 \u0026ndash; Human alveolar type II cell marker 280\u003c/p\u003e\n \u003cp\u003eIF \u0026ndash; Immunofluorescence\u003c/p\u003e\n \u003cp\u003eILD \u0026ndash; Interstitial lung disease\u003c/p\u003e\n \u003cp\u003eIPF \u0026ndash; Idiopathic pulmonary fibrosis\u003c/p\u003e\n \u003cp\u003eiPSC \u0026ndash; Induced pluripotent stem cell\u003c/p\u003e\n \u003cp\u003eKRT5 \u0026ndash; Keratin 5\u003c/p\u003e\n \u003cp\u003eLB \u0026ndash; Lamellar body\u003c/p\u003e\n \u003cp\u003eLDH \u0026ndash; Lactate dehydrogenase\u003c/p\u003e\n \u003cp\u003eLDHA \u0026ndash; Lactate dehydrogenase A\u003c/p\u003e\n \u003cp\u003eLAMP3 \u0026ndash; Lysosomal-associated membrane protein 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 40.1717%;\"\u003e\n \u003cp\u003eMACS \u0026ndash; Magnetic-activated cell sorting\u003c/p\u003e\n \u003cp\u003eMHH \u0026ndash; Medizinische Hochschule Hannover\u003c/p\u003e\n \u003cp\u003eMS \u0026ndash; Mouse antibody (context: anti-HT-II-280 ms monoclonal\u003c/p\u003e\n \u003cp\u003eMT \u0026ndash; Mitochondria\u003c/p\u003e\n \u003cp\u003eMV \u0026ndash; Microvilli\u003c/p\u003e\n \u003cp\u003eNC \u0026ndash; Nucleus\u003c/p\u003e\n \u003cp\u003eNKX2-1 / TTF-1 \u0026ndash; NK2 homeobox 1 / Thyroid transcription factor-1\u003c/p\u003e\n \u003cp\u003eOCR \u0026ndash; Oxygen consumption rate\u003c/p\u003e\n \u003cp\u003eP \u0026ndash; Passage\u003c/p\u003e\n \u003cp\u003ePDK1 \u0026ndash; Pyruvate dehydrogenase kinase 1\u003c/p\u003e\n \u003cp\u003ePKM2 \u0026ndash; Pyruvate kinase isoform M2\u003c/p\u003e\n \u003cp\u003eproSP-C \u0026ndash; Pro-surfactant protein C\u003c/p\u003e\n \u003cp\u003eRB \u0026ndash; Rabbit antibody (context: anti-proSP-C rb polyclonal)\u003c/p\u003e\n \u003cp\u003eRotAA \u0026ndash; Rotenone \u0026amp; antimycin A\u003c/p\u003e\n \u003cp\u003eSFTPB \u0026ndash; Surfactant protein B\u003c/p\u003e\n \u003cp\u003eSFTPC / SP-C \u0026ndash; Surfactant protein C\u003c/p\u003e\n \u003cp\u003eTEM \u0026ndash; Transmission electron microscopy\u003c/p\u003e\n \u003cp\u003eTGF-\u0026beta; \u0026ndash; Transforming growth factor beta\u003c/p\u003e\n \u003cp\u003eTTF-1 \u0026ndash; Thyroid transcription factor-1 (NKX2-1)\u003c/p\u003e\n \u003cp\u003eWST-1 \u0026ndash; Water-soluble tetrazolium-1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003ch2\u003eEthics statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by the Ethic Committee of MHH (ethic votes 8867_BO_K_2020 and 10194_BO_K_2022) and followed the rules of the Declaration of Helsinki of 1975. All patients provided written informed consent to participate in this study.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eIdea and conceptualization: LJS, JR, SSCH, JCK, LN\u003c/p\u003e\n\u003cp\u003eFunding acquisition: JCK, LN, MMH\u003c/p\u003e\n\u003cp\u003eClinical diagnosis validation and human tissue acquisition: CW, MK, RE, CPM, FI, PZ\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: LJS, JR, SMJ, MK, RE, CPM\u003c/p\u003e\n\u003cp\u003eData acquisition: LJS, JR, LK\u003c/p\u003e\n\u003cp\u003eData analysis and interpretation: LJS, JR\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: LJS\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review: All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003ch2\u003eFunding\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study was funded by the German Center for Lung Research (Deutsches Zentrum f\u0026uuml;r Lungenforschung, DZL) and the Else-Kr\u0026ouml;ner-Fresenius Foundation.\u003c/p\u003e\n\u003ch2\u003eCompeting interest\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLederer DJ, Martinez FJ. 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Contributions of alveolar epithelial cell quality control to pulmonary fibrosis. J Clin Invest. 2020;130:5088\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003evan Riet S, van Schadewijk A, Khedoe PPSJ, Limpens RWAL, B\u0026aacute;rcena M, Stolk J, Hiemstra PS, van der Does AM. Organoid-based expansion of patient-derived primary alveolar type 2 cells for establishment of alveolus epithelial Lung-Chip cultures. Am J Physiol Lung Cell Mol Physiol. 2022;322:L526\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuezawa T, Kanagaki S, Moriguchi K, Masui A, Nakao K, Toyomoto M, Tamai K, Mikawa R, Hirai T, Murakami K, Hagiwara M, Gotoh S. Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids. Stem Cell Rep. 2021;16:2973\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"alveolar type II cells, lung epithelium, lung organoids, alveolosphere, Idiopathic pulmonary fibrosis, human lung explant, fibrotic end-stage","lastPublishedDoi":"10.21203/rs.3.rs-7913465/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7913465/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II identity, enable cryopreservation, and capture disease-specific metabolic alterations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHT-II-280\u003csup\u003e+\u003c/sup\u003e AT-II cells were isolated by magnetic bead sorting from 62 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 21 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II identity was verified by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after \u0026ge;\u0026thinsp;28 days and tested for viability. Metabolic profiling was performed using extracellular flux assays.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAT-II cells were successfully (~\u0026thinsp;80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (\u0026gt;\u0026thinsp;90% in P1), especially in AT-II cells from fibrotic explants. Interestingly, primary tissue-derived lung organoids display heterogeneous morphologies and sizes, particularly in fibrotic-derived cultures indicated by histology and microcomputed tomography. Culturing conditions were optimized to avoid differentiation towards AT-I cells or aberrant basaloid cells. Lineage fidelity was preserved across passages, with stable expression of proSP-C, HT-II-280, and pronounced presence of lamellar bodies. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, highlighting possibility for on demand long-term storage. Fibrotic organoids exhibited metabolic reprogramming illustrated by a pronounced glycolytic shift with increased ATP production.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe established a robust and reproducible cell-line-free 3D platform from primary human AT-II cells of end-stage ILD lungs to generate personalized lung organoids. These organoids retain AT-II identity across passages, remain viable after cryostorage, and recapitulate patient-specific metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting possible mitochondrial dysfunction and high energy demand. This reproducible scalable model provides a transferable resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for precision medicine.\u003c/p\u003e","manuscriptTitle":"Human Alveolar Type II Organoids from Fibrotic Lungs Capture Disease-Specific Metabolic Reprogramming and Provide a Platform for Personalized Medicine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 13:53:14","doi":"10.21203/rs.3.rs-7913465/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-25T15:13:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T05:42:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T01:49:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95626946068665461828594199907652680164","date":"2025-10-24T23:08:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315546180617954951531852283539016743730","date":"2025-10-23T19:13:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T17:00:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-23T08:30:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-23T07:55:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Respiratory Research","date":"2025-10-21T10:19:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"505dfc53-bee7-4fc6-9239-4085cdf6339d","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:13:11+00:00","versionOfRecord":{"articleIdentity":"rs-7913465","link":"https://doi.org/10.1186/s12931-026-03610-9","journal":{"identity":"respiratory-research","isVorOnly":false,"title":"Respiratory Research"},"publishedOn":"2026-03-07 15:57:09","publishedOnDateReadable":"March 7th, 2026"},"versionCreatedAt":"2025-11-04 13:53:14","video":"","vorDoi":"10.1186/s12931-026-03610-9","vorDoiUrl":"https://doi.org/10.1186/s12931-026-03610-9","workflowStages":[]},"version":"v1","identity":"rs-7913465","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7913465","identity":"rs-7913465","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}