PuSH - Publikationsserver des Helmholtz Zentrums München: Cold Storage of Human Precision-cut Lung Slices Preserves Cellular and Transcriptional Identity Enabling Optimized On-demand Translational Lung Research.

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Narvaez, M.M.* ; Gölitz, F.* ; Jain, E. ; Gote-Schniering, J.* ; Stoleriu, M.G.* ; Bertrams, W.* ; Schmeck, B.* ; Yildirim, A.Ö. ; Rauen, U.* ; Wille, T.* ; Lehmann, M.*

Cold Storage of Human Precision-cut Lung Slices Preserves Cellular and Transcriptional Identity Enabling Optimized On-demand Translational Lung Research.

Am. J. Respir. Crit. Care Med. 211, A7646 - A7646 (2025)

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RATIONALE: Human precision-cut lung slices (hPCLS) are a unique platform for functional, mechanistic, and drug discovery studies in respiratory research. Their relevance lies in their ability to maintain all resident cellular compartments (epithelial, mesenchymal, and immune cells) as well as the extracellular matrix (ECM) in their native three-dimensional structure. However, tissue availability, transportation, generation, and cultivation time represent important challenges for their usage. To address this, the present study aimed to evaluate the efficacy of a specifically designed tissue preservation solution (TiProtec) in the absence (-) or presence (+) of iron chelators as an alternative for long-term cold storage of hPCLS. METHODS: 500 µm hPCLS were generated and stored either in DMEM/F-12 medium or TiProtec (-/+) for up to 28 days. Viability, metabolic activity, and tissue structure were longitudinally determined. Bulk-RNA sequencing was used to study transcriptional changes, regulated signaling pathways, and changes in cellular composition after cold storage. Moreover, the induction of cold storage-associated cellular senescence was determined by transcriptomics and immunofluorescence (IF). To evaluate their potential for mechanistic studies in lung research, we evaluated the response to a previously described fibrotic cocktail after 7 and 14 days of cold storage in TiProtec (-/+) by IF and RT-qPCR. RESULTS: We demonstrated that TiProtec (+) preserves the viability, metabolic activity, transcriptional profile, and cellular composition of hPCLS for up to 14 days when compared to freshly sliced hPCLS. Moreover, cold storage did not significantly induce cellular senescence in hPCLS. Notably, TiProtec (+) downregulated pathways associated with cell death and inflammation while activating pathways protective against oxidative stress. Finally, cold-stored hPCLS remained responsive for up to 14 days to a fibrotic cocktail upregulating the expression of fibrosis-associated proteins such as fibronectin, alpha-smooth muscle actin, and alpha-1 type I collagen. CONCLUSION: This study provides for the first time insights into the transcriptional and functional changes associated with cold storage preservation of hPCLS. Moreover, it contributes to an optimized use of hPCLS, enabling banking, sharing, and on-demand processing and usage of hPCLS for translational lung research.

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