47The complexity of the lung microenvironment together with changes in cellular 48 composition during disease progression make it exceptionally hard to understand the 49 molecular mechanisms leading to the development of chronic lung diseases. Although 50 recent advances in cell type resolved and single-cell sequencing approaches hold 51 great promise for studying complex diseases, their implementation greatly relies on 52 local access to fresh tissue, as traditional methods to process and store tissue do not 53 allow viable cell isolation. To overcome these hurdles, we developed a novel, versatile 54 workflow that allows long-term storage of human lung tissue with high cell viability, 55 permits thorough sample quality check before cell isolation, and is compatible with 56 next generation sequencing-based profiling, including single-cell approaches. We 57 demonstrate that cryopreservation is suitable for isolation of multiple cell types from 58 different lung locations and is applicable to both healthy and diseased tissue, including 59 COPD and tumor samples. Basal cells isolated from cryopreserved airways retain the 60 ability to differentiate, indicating that cellular identity is not altered by cryopreservation. 61 Importantly, using RNA sequencing (RNA-seq) and Illumina EPIC Array, we show that 62 genome-wide gene expression and DNA methylation signatures are preserved upon 63 cryopreservation, emphasizing the suitability of our workflow for -omics profiling of 64 human lung cells. In addition, we obtained high-quality single-cell RNA sequencing 65 data of cells isolated from cryopreserved human lung, demonstrating that 66 cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, 67 3 our cryopreservation workflow is well-suited for prospective tissue collection by 68 academic collaborators and biobanks, opening worldwide access to human tissue. 69 70
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