Cold storage of human precision-cut lung slices in TiProtec preserves cellular composition and transcriptional responses and enables on-demand mechanistic studies.

Mareike Lehmann, Ursula Rauen, Bernd Schmeck, Eshita Jain, Ali Önder Yildirim, Mircea Gabriel Stoleriu, M Camila Melo-Narvaez, Fee Gölitz, Janine Gote-Schniering, Wilhelm Bertrams, Timo Wille

Journal: Respiratory research 2025;26(1):57

PMID: 39962456

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Abstract

BACKGROUND

Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in the field of respiratory research. However, tissue availability, generation, and cultivation time represent important challenges for their usage. Therefore, the present study evaluated the efficacy of a specifically designed tissue preservation solution, TiProtec, complete or in absence (-) of iron chelators, for long-term cold storage of hPCLS.

METHODS

hPCLS were generated from peritumor control tissues and stored in DMEM/F-12, TiProtec, or TiProtec (-) for up to 28 days. Viability, metabolic activity, and tissue structure were determined. Moreover, bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and cellular composition after cold storage. Induction of cold storage-associated senescence was determined by transcriptomics and immunofluorescence (IF). Finally, cold-stored hPCLS were exposed to a fibrotic cocktail and early fibrotic changes were assessed by RT-qPCR and IF.

RESULTS

Here, we found that TiProtec preserves the viability, metabolic activity, transcriptional profile, as well as cellular composition of hPCLS for up to 14 days. Cold storage did not significantly induce cellular senescence in hPCLS. Moreover, TiProtec downregulated pathways associated with cell death, inflammation, and hypoxia while activating pathways protective against oxidative stress. Cold-stored hPCLS remained responsive to fibrotic stimuli and upregulated extracellular matrix-related genes such as fibronectin and collagen 1 as well as alpha-smooth muscle actin, a marker for myofibroblasts.

CONCLUSIONS

Optimized long-term cold storage of hPCLS preserves their viability, metabolic activity, transcriptional profile, and cellular composition for up to 14 days, specifically in TiProtec. Finally, our study demonstrated that cold-stored hPCLS can be used for on-demand mechanistic studies relevant for respiratory research.

Address: Comprehensive Pneumology Center with the CPC-M bioArchive and Institute of Lung Health and Immunity, Helmholtz Center Munich, German Center for Lung Research (DZL), Munich, Germany.; Institute for Lung Research, Philipps-University Marburg, German Center for Lung Research (DZL), Marburg, Germany.; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany.; Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Munich, Germany.; Comprehensive Pneumology Center with the CPC-M bioArchive and Institute of Lung Health and Immunity, Helmholtz Center Munich, German Center for Lung Research (DZL), Munich, Germany.; Department of Rheumatology and Immunology, Department of Pulmonary Medicine, Allergology and Clinical Immunology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.; Lung Precision Medicine (LPM), Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland.; Comprehensive Pneumology Center with the CPC-M bioArchive and Institute of Lung Health and Immunity, Helmholtz Center Munich, German Center for Lung Research (DZL), Munich, Germany.; Division for Thoracic Surgery Munich, Ludwig-Maximilians-University of Munich (LMU) and Asklepios Lung Clinic Munich-Gauting, Gauting, Germany.; Institute for Lung Research, Philipps-University Marburg, German Center for Lung Research (DZL), Marburg, Germany.; Institute for Lung Research, Philipps-University Marburg, German Center for Lung Research (DZL), Marburg, Germany.; Core Facility Flow Cytometry - Bacterial Vesicles, Philipps-University Marburg, Marburg, Germany.; Department of Medicine, Pulmonary and Critical Care Medicine, University Medical Center Marburg, Philipps-University Marburg, German Center for Lung Research (DZL), Marburg, Germany.; Center for Synthetic Microbiology (Synmikro), Philipps-University Marburg, Marburg, Germany.; Member of the German Center of Infectious Disease Research, Marburg, Germany.; Institute for Lung Health (ILH), German Center for Lung Research (DZL), Giessen, Germany.; Comprehensive Pneumology Center with the CPC-M bioArchive and Institute of Lung Health and Immunity, Helmholtz Center Munich, German Center for Lung Research (DZL), Munich, Germany.; Institute of Experimental Pneumology (IEP), Ludwig-Maximilians University of Munich (LMU), Munich, Germany.; Institute of Physiological Chemistry, University Hospital Essen, Essen, Germany.; Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany. [email protected].; Department of CBRN Medical Defense, Bundeswehr Medical Academy, Munich, Germany. [email protected].; Comprehensive Pneumology Center with the CPC-M bioArchive and Institute of Lung Health and Immunity, Helmholtz Center Munich, German Center for Lung Research (DZL), Munich, Germany. [email protected].; Institute for Lung Research, Philipps-University Marburg, German Center for Lung Research (DZL), Marburg, Germany. [email protected].; Institute for Lung Health (ILH), German Center for Lung Research (DZL), Giessen, Germany. [email protected].

DOI: 10.1186/s12931-025-03132-w

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https://assays.cancer.gov/non-CPTAC-1113

MeSH Terms: Humans, Lung, Cryopreservation, Transcription, Genetic, Organ Preservation Solutions