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

IF 5.8 2区医学 Q1 Medicine Respiratory Research Pub Date : 2025-02-17 DOI:10.1186/s12931-025-03132-w

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

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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.

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背景：人体精密切片肺片（hPCLS）是呼吸研究领域进行功能、机理和药物发现研究的独特平台。然而，组织的可用性、生成和培养时间是其使用面临的重要挑战。因此，本研究评估了专门设计的组织保存溶液 TiProtec（含铁螯合剂或不含（-）铁螯合剂）在长期冷藏 hPCLS 方面的功效。方法：hPCLS 由肿瘤周围对照组织生成，在 DMEM/F-12、TiProtec 或 TiProtec（-）中保存长达 28 天。对其存活率、代谢活性和组织结构进行测定。此外，还使用大量 RNA 测序来研究冷藏后的转录变化、调控信号通路和细胞组成。通过转录组学和免疫荧光（IF）测定了冷藏相关衰老的诱导。最后，将冷藏的 hPCLS 暴露于纤维化鸡尾酒中，并通过 RT-qPCR 和 IF 评估早期纤维化变化：结果：我们发现 TiProtec 能保持 hPCLS 的活力、代谢活性、转录谱以及细胞组成长达 14 天。冷藏并没有明显诱导 hPCLS 的细胞衰老。此外，TiProtec 下调了与细胞死亡、炎症和缺氧相关的通路，同时激活了保护细胞免受氧化应激的通路。冷藏的 hPCLS 对纤维化刺激仍有反应，并上调细胞外基质相关基因，如纤维粘连蛋白和胶原 1 以及肌成纤维细胞的标志物--α-平滑肌肌动蛋白：经过优化的 hPCLS 长期冷藏可保持其活力、代谢活性、转录谱和细胞组成长达 14 天，特别是在 TiProtec 中。最后，我们的研究表明，冷藏的 hPCLS 可用于与呼吸研究相关的按需机理研究。

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来源期刊

Respiratory Research RESPIRATORY SYSTEM-

CiteScore

9.70

自引率

1.70%

发文量

314

审稿时长

4-8 weeks

期刊介绍： Respiratory Research publishes high-quality clinical and basic research, review and commentary articles on all aspects of respiratory medicine and related diseases. As the leading fully open access journal in the field, Respiratory Research provides an essential resource for pulmonologists, allergists, immunologists and other physicians, researchers, healthcare workers and medical students with worldwide dissemination of articles resulting in high visibility and generating international discussion. Topics of specific interest include asthma, chronic obstructive pulmonary disease, cystic fibrosis, genetics, infectious diseases, interstitial lung diseases, lung development, lung tumors, occupational and environmental factors, pulmonary circulation, pulmonary pharmacology and therapeutics, respiratory immunology, respiratory physiology, and sleep-related respiratory problems.