Background:The extracellular matrix (ECM) is a key regulator of tissue homeostasis and remodeling in interstitial lung diseases (ILD) such as idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (FHP). Decellularized lung scaffolds provide a physiologically relevant platform for studying cell-matrix interactions but generating structurally intact scaffolds from fibrotic human lungs remains challenging. Many existing protocols struggle with either insufficient removal of the cells or significant disruption of certain ECM components. In addition, all of them rely on agarose-embedding for the production of thin lung slices. Methods:Tissue samples were decellularized using apoptosis inducers (Camptothecin, Raptinal) and a mild detergent (SB-10). Standardized decellularized lung scaffolds (SDLS) were generated using a gelatin-based temporary cutting support structure, which is completely removable prior to recellularization. Decellularization and preservation of the ECM was validated by DAPI nuclear counts and semi-quantitative image analysis of collagen (picro-sirius red under polarization) and elastin (elastic Verhoeff-Van Gieson (EVG)). For functional testing, SDLS were repopulated with primary human lung fibroblasts (CD90+CD31-CD45-) and assessed for colonization, viability (WST-1 assay), and cytotoxicity (LDH assay). Results:The protocol removed all nuclei (>99.8%) in both fibrotic and non-fibrotic lung tissue while preserving structural integrity and the major ECM components (collagen and elastin). The novel semi-quantitative analysis proved to be accurate and versatile for the analysis of multiple ECM components within one sample. Overall, both Collagen (fresh 15.5% ± 4.3 vs. decellularized 16.7% ± 4.4) and elastin (fresh 10.0% ± 3.7 vs. decellularized 9.8% ± 3.4) remained stable post-decellularization. Fibroblasts successfully colonized SDLS, with non-fibrotic scaffolds showing pronounced contraction, whereas fibrotic scaffolds retained their dimensions. Even though fibroblasts seeded on SDLS showed significantly lower viability than those seeded on plastic plates, cytotoxicity 1 day after repopulation was not increased and remained acceptable. Conclusion:We present a reproducible, agarose-free, apoptosis-assisted method for the generation of residue-free and standardized decellularized lung scaffolds from normal and fibrotic human tissue. The novel semi-quantitative histological analysis allows the investigation of multiple ECM components from a single sample. The SDLS platform preserves key ECM components and supports controlled recellularization enabling physiologically relevant studies of matrix-cell interactions and therapeutic screening in ILD models.

RATIONALE:Restrictive allograft syndrome (RAS) is a major cause of mortality following lung transplantation due to progressive fibrosis of the lung allograft with no therapeutic options. Knowledge of the cellular and molecular mechanisms driving fibrosis in RAS remains limited. OBJECTIVE:To characterize the cellular and molecular changes in human RAS lungs through single-cell transcriptomic profiling. METHODS:Single-nucleus RNA-sequencing (snRNA-seq) was performed in peripheral lung tissues from 15 RAS patients undergoing lung re-transplantation, and from 9 healthy control lungs. Findings were validated and extended using histologic techniques including immunofluorescence, RNA in situ hybridization, Elastica-van-Gieson immunohistochemistry, quantitative histological analyses, and micro-CT scans. MEASUREMENTS AND MAIN RESULTS:snRNA-seq analysis of RAS lungs revealed previously undescribed aberrant basaloid cells, ectopic COL15A1+ peribronchial vascular endothelial cells (pVECs), and CTHRC1+ fibrotic fibroblasts. Histologic stains disclosed distinctive distribution patterns: aberrant basaloid cells, primarily localized at the fibrotic edge, together with juxtaposed CTHRC1+ fibrotic fibroblasts and ectopic COL15A1+ pVECs form the fibrotic niche of alveolar fibroelastosis (AFE). PRX+ alveolar microvasculature is partially lost in AFE areas. Micro-CT scans revealed changes from pulmonary to systemic perfusion, facilitated by COL15A1+ pVECs. Last, our data reveals potential therapeutic targets in RAS, including integrin αvβ6, activator of TGFβ. CONCLUSION:Considering the multifaceted differences of RAS and idiopathic pulmonary fibrosis, we revealed a surprising general principle of an entity-spanning composition of the fibrotic niche by aberrant basaloid cells localized at the fibrotic edge, ectopic COL15A1+ pVECs and CTHRC1+ fibrotic fibroblasts. This suggests a flexible but cellular pathogenesis-guided transferability of potential therapeutic approaches between progressive fibrotic lung diseases.

Ultra-high-resolution propagation-based synchrotron phase-contrast CT is an emerging technique for lung imaging. However, its feasibility and diagnostic potential at radiation doses comparable to those used in standard clinical procedures has yet to be established. This study aims to evaluate the performance of phase-contrast CT in comparison with state-of-the-art high-resolution multislice CT and bronchoscopy, and to validate its diagnostic accuracy histologically using porcine and, for the first time, human lung specimens. Phase-contrast CT experiments were conducted at the Italian synchrotron using lung specimens mounted in a custom-made anthropomorphic chest phantom. Imaging utilized two photon-counting detectors under various acquisition settings, followed by artificial intelligence-based denoising. Sequential imaging by phase-contrast CT, multislice CT, and bronchoscopy was performed prior to formaldehyde vapor fixation and histological dissection. Image quality was assessed quantitatively (contrast-to-noise ratio, edge sharpness, power spectra) and qualitatively via radiological scoring across 14 criteria. Phase-contrast CT achieved effective pixel sizes of 0.067 mm (Hydra detector) and 0.038 mm (LAMBDA detector), at radiation doses near full-dose multislice CT (≈ 12 mGy). Denoising improved contrast without major loss of edge sharpness. Radiological scoring showed phase-contrast CT outperformed multislice CT in visualizing peripheral airways and fine parenchymal structures. Histological validation confirmed imaging accuracy. Limitations from source spot size (≈ 200 μm) were noted but did not prevent significant diagnostic improvements. Phase-contrast CT, combined with artificial intelligence-based denoising, offers detailed, non-invasive imaging of lung microstructures at clinically relevant radiation doses. It complements multislice CT, holds potential for clinical adoption in advanced pulmonary diagnostics, and may reduce reliance on invasive biopsies.

Precision-cut lung slices (PCLS) are a complex three-dimensional ex vivo model system comprised of all resident cell types of the lung, thus closely mimicking the in vivo situation in regards to structural composition and function. The herein described application of a precise airway epithelial lesion via femtosecond laser-based nanosurgery and subsequent longitudinal imaging via two-photon or confocal microscopy enables the examination of the tissue's repair responses on a single-cell level. Allowing for live observation of intercellular cross-talk, this study demonstrates an endogenous repair program is induced in human PCLS upon damage induction. As early reaction to a small epithelial lesion, physiological stress responses, including transient airway constriction and increased mucus secretion, occur, followed by epithelial restitution within 24 h. Automated cell detection and subsequent cell track analysis reveal a more linearly confined cellular movement in the course of repair. Further, non-stationary, motile cells directly interact with cell debris, thereby contributing to final resolution of the lesion. Together, these findings emphasize the suitability of PCLS, combined with localized laser-based damage induction and state-of-the-art microscopy techniques, as a model system to study complex intercellular interactions in the course of endogenous repair processes.

BACKGROUND:Cardiomyocyte mitochondria align with sarcomeres during heart development. Mitochondrial motility is controlled by RHOT (ras homolog family member T) 1 and RHOT2. RHOT1 and RHOT2 are atypical Rho-like small GTPases that are anchored to the outer mitochondrial membrane and couple mitochondria to kinesin and dynein motors. We hypothesized that RHOT protein expression and mitochondrial motility are required for mitochondrial positioning during cardiomyocyte development.METHODS:We generated mice with cardiomyocyte-selective deletion of Rhot1 and Rhot2 during embryogenesis (cRhot1/2-KO [constitutive and embryonic cardiomyocyte-selective Rhot1/2 knockout]) or tamoxifen-inducible deletion in the adult heart (iRhot1/2-KO [inducible cardiomyocyte-selective Rhot1/2 knockout]) to assess the importance of mitochondrial motility during and after cardiomyocyte maturation. Mitochondrial motility was determined by a motor protein-driven single mitochondria motility assay. Respiratory capacity was measured in isolated mitochondria. Intracellular mitochondrial localization and ATP production in isolated cardiomyocytes were assessed by confocal microscopy and after adenoviral expression of the fluorescence resonance energy transfer-based ATP biosensor ATeam. Cardiac ultrastructure was assessed by electron micrographs; mass spectrometry was used for proteome analysis.RESULTS:cRhot1/2-KO mice developed fatal cardiomyopathy associated with sarcomere disarray and perinuclear accumulation of mitochondria and ATP production. Mitochondria isolated from cRhot1/2-KO hearts exhibited impaired motility but preserved respiratory capacity. Mechanistically, proteome analysis identified that RHOT proteins bind mitochondria to contractile muscle fiber proteins. In contrast, inducible deletion of Rhot1 and Rhot2 in adult iRhot1/2-KO mice did not result in heart failure. Despite impaired motility of isolated mitochondria, intracellular mitochondrial localization, local ATP production, and sarcomere structure were preserved in adult iRhot1/2-KO hearts after cardiomyocyte maturation.CONCLUSIONS:RHOT proteins bind mitochondria to contractile muscle fiber proteins and are required for mitochondrial positioning in cardiomyocytes during development. Our study links mitochondrial motility and local ATP production to structural and functional maturation of the heart.

ObjectiveThe objective of this study was to gain insight into the molecular mechanisms leading to early degeneration of decellularized homografts.MethodsFormalin-fixed and paraffin-embedded tissues from fresh explanted decellularized aortic (n = 7) and pulmonary (n = 8) valves were used. RNA was isolated and analyzed using panel-based transcriptomics, focusing on fibrosis- and inflammation-related genes. Differentially expressed genes were used as input parameters for biological pathway analysis using the Gene Ontology Biological Process and Hallmark databanks. Formalin-fixed and paraffin-embedded tissues from freshly explanted healthy donor aortic (n = 7) and pulmonary valves (n = 8) were used as controls.ResultsOur analysis revealed 56 differentially expressed genes in decellularized aortic valves compared to donor aortic valves, of which 21 and 35 were up- and downregulated, respectively. Decellularized pulmonary valves showed 115 differentially expressed genes compared to donor pulmonary valves, 66 of which were up- and 49 downregulated. In both decellularized aortic and pulmonary valve explants, we found increased expression of fibrosis-, inflammation-, and endothelium-related genes and a decreased expression of genes encoding for complement factors in line with the observed biological pathway activity patterns.ConclusionWe present a comprehensive transcriptome analysis of explanted decellularized heart valve homografts providing insights into the biological processes leading to continued degeneration and ultimately loss of function. The degeneration of decellularized homografts is driven by inflammation, fibrosis, and extracellular matrix (ECM) remodelling, reinforced by ongoing oxidative stress rather than by a mainly complement-driven humoral immune response.

Abstract This study demonstrates the successful production and injection of human induced pluripotent stem cell cardiomyocyte aggregates into infarcted cynomolgus monkey hearts, resulting in substantial, structured human grafts three months after cell transplantation. Transient graft-induced arrhythmias decreased over time. Both the arrhythmogenicity and the substantial heart function recovery in vivo notably seemed to correlate with induced pluripotent stem cell clone-dependent contractile and electrophysiological cardiomyocyte properties in vitro. Overexpression of a red fluorescent reporter protein led to a dysregulated conduction and contraction machinery in yet engraftment competent cardiomyocytes, providing an important tool to mechanistically understand and improve induced pluripotent stem cell-based heart repair in preclinical models. We demonstrate the logistically important, temporal uncoupling of cardiomyocyte production from transplantation. Cardiomyocyte aggregate transplantation yielded results comparable to the reported transplantation of 10-20-fold higher numbers of dissociated human embryonic stem cell- cardiomyocytes and suggests a higher degree of cell/ tissue maturation in cardiac grafts. Our study promotes reduced cell production costs, highlights the need for an in vitro potency assay, and shows a pragmatic new avenue for the clinical translation of human induced pluripotent stem cell-based heart repair.

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-associated features, enable cryopreservation, and capture disease-associated metabolic alterations. Methods HT-II-280+ AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. 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. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production. Conclusion We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.

Pregnancy-associated hemodynamic overload and hormonal changes induce hypertrophy and metabolic remodeling of the maternal heart. Mitochondrial motility, mediated by ras homolog family member T (RHOT) 1 and RHOT2, is essential for cardiac adaptation to increased workload, cardiomyocyte hypertrophy, and sarcomere maturation. To test the hypothesis that Rhot1/2 expression is required for pregnancy- and postpartum-associated adaptations of the maternal heart, female mice with tamoxifen-inducible, cardiomyocyte-selective deletion of Rhot1 and Rhot2 (iRhot1/2-KO) were mated. Following gene deletion in adult mice, cardiac tissue and function were analyzed after three to five successive pregnancies and postpartum nursing periods. Age-matched nulliparous iRhot1/2-KO mice and age-matched mice expressing Rhot1 and Rhot2 served as controls. Motility of mitochondria isolated from iRhot1/2-KO hearts was impaired, as determined by the number of mobile mitochondria in an in vitro motor protein-driven single mitochondrion motility assay performed on surface-immobilized microtubules. Despite loss of Rhot1/2 expression, contractile function assessed by transthoracic echocardiography, mRNA expression of peripartum-associated heart failure markers, cardiac structure, mitochondrial morphology, mitochondrial enzymatic activity, and mitochondrial DNA content were all comparable to controls expressing Rhot1/2 at the investigated time points. RNA sequencing-based gene profiling identified a transcriptional program through which RHOT proteins preserve cardiac energetic and contraction gene expression during pregnancy and postpartum. Together, cardiomyocyte-selective loss of Rhot1/2 expression in the adult heart does not cause peripartum-associated heart failure, despite reduced cardiac energetic and contraction gene expression.

Pleuroparenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease (ILD) with defining histology of intra-alveolar fibrosis with septal elastosis (AFE), suggesting unique cellular disease processes. Here, we present a binational single-nucleus RNA sequencing atlas of PPFE, based on explanted lungs from 40 patients. Immunofluorescence microscopy, RNA in situ hybridization, micro-computed tomography (CT), and hierarchical phase-contrast (HiP) synchrotron CT provided spatial context. We identify PPFE-associated adventitial and elastofibrotic fibroblasts as key drivers of elastotic remodeling within an inflammatory microenvironment, maintained by immune cells forming tertiary lymphoid structures. Spatial mapping reveals an intriguing zonation of AFE, maintained by intercellular circuits between PPFE-associated cell types. Comparative analysis with idiopathic pulmonary fibrosis highlights CTHRC1+ fibrotic fibroblasts and aberrant basaloid cells as conserved profibrotic cellular machinery mediating collagen deposition across ILDs. This integrative atlas defines the cellular landscape of PPFE and dissects elastotic from fibrotic remodeling, providing a molecular rationale for niche-specific therapeutic strategies.

Abstract Background Chronic lung allograft dysfunction (CLAD) is the leading cause of death beyond the first year after lung transplantation, and its most frequent phenotype is bronchiolitis obliterans syndrome (BOS), a fibrotic small-airway disease. Mechanistic work has focused on the immune compartment, yet intensified immunosuppression does not alter established disease. Aim To resolve which structural cell states populate the BOS graft and how they are spatially organized during airway obliteration. Methods We profiled explanted lungs from 33 BOS patients undergoing re-transplantation and 33 controls, combining single-nucleus RNA sequencing (14 BOS, 13 controls) with targeted spatial transcriptomics of 108 regions (27 BOS, 24 controls) and multiplex immunofluorescence validation. Single-nucleus data were integrated with a published restrictive allograft syndrome (RAS) atlas. Results Across 175,128 nuclei and 1.67 million spatially resolved cells, BOS lungs harbored a profibrotic circuit of Aberrant Basaloid cells and CTHRC1 + fibrotic fibroblasts previously described in fibrotic lung diseases, including RAS. Spatial mapping identified a CXCL14 + TNC + injury-associated basal cell state arising early in the obliterative cascade, identifying basal cells as their major reservoir. CTHRC1 + fibroblasts expanded subepithelially replacing resident peribronchial fibroblasts, alongside a peribronchial vascular shift toward systemic venous endothelium. The circuit extended beyond the airway wall to the alveolar interface, defining two convergent remodeling fronts. Conclusion BOS engages structural-cell circuits largely shared with RAS and fibrotic lung diseases, but along an airway-centered rather than parenchyma-centered axis. CLAD thus emerges as a spatial rather than cellular spectrum, defined by anatomical distribution more than cell identity. Shared structural programs may therefore be targetable across CLAD phenotypes.

BACKGROUND:Long-term survival after lung transplantation (LTx) remains highly variable, with chronic lung allograft dysfunction (CLAD) as a major limiting factor. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS) in more than 50% of LTx recipients. In contrast, a subgroup of "super survivors" maintains long-term graft stability for years without immunological complications. These patients show an increased prevalence of alveolar macrophages (AMs), but the mechanisms underlying stable graft function remain unclear. METHODS:Transcriptome profiles of AMs were analyzed in lung tissues of super survivors (n = 15), recipients with BOS or RAS (n = 24), and healthy controls (n = 9) using spatial transcriptomics. AM origin was assessed in sex-mismatched cases (n = 8) using X/Y fluorescence in situ hybridization. RESULTS:In super survivor AMs, upregulated genes were associated with stress control and detoxification (GSTA2, HBA2), innate immune regulation (INAVA), lipid homeostasis (APOE, CES1), and alveolar structure maintenance. Most AMs were donor-derived (61.3%). The majority (78.26%) displayed a pre-activated state with enhanced immune plasticity, while a smaller fraction (13.04%) showed M2-like repair functions. In BOS and RAS lungs, donor-derived AMs (37.4%) were largely replaced by recipient-derived cells. 35.71% of BOS and 56.52% of RAS AMs exhibited a progressive M1-like polarization. Transitional pre-BOS and pre-RAS stages suggested that early post-transplant conditions shape macrophage polarization and influence long-term outcomes. CONCLUSIONS:Stable long-term graft function after LTx is associated with persistence of metabolically adapted donor-derived AMs, whereas CLAD reflects their replacement by inflammatory recipient cells. Preserving protective macrophage populations may help promote a long-term stable immune microenvironment after LTx.

IntroductionIdiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease with high mortality. Current therapies are very limited, with nintedanib and pirfenidone being the only non-invasive but non-curative interventions, ultimately bridging to lung transplantation.MethodsIn silico modeling of dysregulated pathways in IPF and screening for putative interfering small molecules identified carvedilol as a promising anti-fibrotic agent. We validated drug-mediated effects on key features of fibroblast activation in functional assays and gene expression analyses in human embryonic lung fibroblasts (MRC-5). Precision-cut lung slices (PCLSs) generated from human lung tissue were assessed for secreted fibrotic markers’ expression.ResultsTreatment with carvedilol reduced metabolic activity, inhibited cell proliferation, and led to decreased migratory activity, as observed in scratch wound assays, in human lung fibroblasts. The functional profile was reflected at the transcriptional level as commonly known fibrotic marker genes, e.g., alpha smooth muscle actin and collagen 1, were robustly repressed. Proteomic profiling underlined a strong extracellular matrix interference with elevated syntheses of several collagen types and various integrins, which play a critical role in pro-fibrotic downstream signaling. Comparison of healthy and fibrotic lung tissue validated an upregulation of pro-fibrotic miR-21 secretion in the ex vivo PCLS model, which remained unchanged upon carvedilol therapy.ConclusionHerein, carvedilol demonstrated significant anti-fibrotic effects on human lung fibroblasts in vitro, thus presenting great potential as an anti-IPF treatment. In addition, miR-21 was validated as a secreted pro-fibrotic biomarker in the ex vivo PCLS model.

Rationale Pulmonary fibrosis (PF) is a rapidly progressive lung disease characterized by uncontrolled deposition of extracellular matrix (ECM) leading to lung stiffening, excessive fibroblast proliferation and destruction of the cellular architecture of the lung. There is currently no curative treatment for PF. Several biomarkers play an important role in the diagnosis and prognosis of PF. The aim of this study is to link molecular biomarkers with mechanical analysis of tissue stiffness in our translational model of human precision-cut lung slices (PCLS). Methods Human PCLS were prepared from PF or non-PF patients and cultured with/without nintedanib or pirfenidone for up to 96 hours. RNA was isolated from the tissues for subsequent NGS sequencing. Both, cytokine responses (ELISA) and microRNA levels (RTqPCR) were assessed in the culture supernatants. Tissue stiffness was measured using the MechanoWell® system. Results Comparing non-fibrotic to fibrotic PCLS after 24 hours in culture, 3117 genes were significantly regulated, of which 1802 were upregulated and 1315 were downregulated in PF tissue. Upregulated genes included ECM-associated matrix metalloproteinases (MMPs) and collagens, which are highly relevant to the progression of lung fibrosis and tissue stiffening. At the protein level, the median levels of pro-collagen I alpha 1 (pCol1a1; 3-fold), MMP9 (43-fold), MMP7 (2-fold) and TGF-β (6-fold) were increased in the PF supernatants compared to non-PF. Furthermore, miR-21 has been reported as a novel biomarker candidate mediating fibrogenic activation of pulmonary fibroblasts and lung fibrosis. We measured 4.1-fold increased levels of soluble miRNA-21 in PF-PCLS vs. non-PF-PCLS. Treatment of fibrotic PCLS with nintedanib and pirfenidone significantly downregulated parenchymal stiffness to 61% and 64%, respectively, compared to control. After 48 hours of cultivation, nintedanib significantly reduced MMP9 (68%), TGF-β (79%) and pCol1a1 (68%), but not MMP7, compared to the medium control. Similar to nintedanib, pirfenidone also downregulated MMP9 (83%). In contrast, pirfenidone treatment led to a significant downregulation of MMP7 (75%) and no significant effect was observed on TGF-β and pCOL1a1. In addition, preliminary experiments showed a tendency for pirfenidone (72%), but not nintedanib, to downregulate microRNA-21. Conclusion Our study combines molecular and mechanical data to assess the efficacy of anti-fibrotic treatments in a fibrotic PCLS model. This approach provides a comprehensive view that can better predict the therapeutic potential of anti-fibrotic drug candidates in early drug development.

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.

The human placenta exhibits a complex three-dimensional (3D) structure with a interpenetrating vascular tree and large internal interfacial area. In a unique and yet insufficiently explored way, this parenchymal structure enables its multiple functions as a respiratory, renal, and gastrointestinal multiorgan. The histopathological states are highly correlated with complications and health issues of mother, and fetus or newborn. Macroscopic and microscopic examination has so far been challenging to reconcile on the entire organ. Here we show that anatomical and histological scales can be bridged with the advent of hierarchical phase-contrast tomography and highly brilliant synchrotron radiation. To this end, we are exploiting the new capabilities offered by the BM18 beamline at ESRF, Grenoble for whole organ as well as the coherence beamline P10 at DESY, Hamburg for high-resolution, creating unique multiscale datasets. We also show that within certain limits, translation to μCT instrumentation for 3D placenta examination becomes possible based on advanced preparation and CT protocols, while segmentation of the datasets by machine learning now remains the biggest challenge.

Resident immune cells are central in shaping the lung’s tissue-specific immunity. Precision-cut lung slices (PCLS) preserve the native tissue microenvironment and are therefore an excellent ex vivo model to analyze residency and functionality of resident memory T cells. To study the modulation of tissue residency markers and T cell activation in the native lung niche, we treated PCLS with broad and T cell-specific stimuli and analyzed responses using flow cytometry and mediator secretion analysis. Using TGFβ, anti-CD3/CD28, IL-2 and a pool of MHC-I restricted peptides we analyzed cytokine secretion, CD4+/CD8+ T cell ratios, and the expression of activation and residency markers. First, we characterized lung immune cell in PCLS which also revealed that resident memory T cells are abundant in PCLS. We showed that regulation of the tissue residency marker CD103 is dependent on TGFβ or IL-2 signaling in combination with T cell receptor engagement. Further, polyclonal activation of T cells in the tissue reduced tissue secretion of anti-inflammatory cytokines like TGFβ, while increasing the secretion of T cell-associated cytokines like IFNγ, IL-2, and Granzyme B. This shift was supported by an upregulation of T cell activation markers such as CD39, CD137, and Ki-67. Finally, treatment of PCLS with a pool of MHC-I-restricted peptides led to increased secretion of multiple inflammatory effector cytokines associated and a specific activation of tissue resident T cells. Taken together, we have demonstrated that PCLS provide an excellent platform to modulate tissue resident T cell responses influenced by human lung tissue microenvironment.