Versatile workflow for cell type–resolved transcriptional and epigenetic profiles from cryopreserved human lung

Abstract

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD phenotypes in human lung fibroblasts

“…1B ). We have previously shown that this step is crucial to ensure exclusion of low-quality control samples presenting additional lung pathologies, which may result in confounding effects in sequencing-based analyse (Llamazares-Prada et al 2021).…”

“…Lung tissue was cryopreserved upon reception as described previously (Llamazares-Prada et al 2021). Briefly, specimens were transported in CO 2 -idepenent medium (Thermo Fisher Scientific) supplemented with 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific) and 1% Amphotericin B (Fisher Scientific).…”

“…As no universal fibroblast markers for FACS are available, primary human lung fibroblasts were isolated by explant outgrowth from tumor-free, distal parenchymal lung tissue that has been depleted from visible airways and vessels, as previously described (Llamazares-Prada et al 2021). Briefly, 7-8 micro-dissected lung parenchyma pieces were placed per well into 6-well plates, let for 30 min at RT without medium to improve explant attachment, and carefully covered with 1 mL of growth medium: DMEM, high glucose, GlutaMAX TM (Thermo Fisher Scientific) supplemented with 2% FBS (Gibco) and 1% penicillin & streptomycin (Thermo Fisher Scientific).…”

“…Cryopreserved lung tissues were thawed for 2 min in a 37ºC water-bath, collected in 50mL Falcon tubes and washed with wash buffer: HBSS supplemented with 2mM EDTA (Thermo Fisher Scientific), 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific) and 1% Amphotericin B (Fisher Scientific). Tissue was minced into smaller pieces prior to mechanical and enzymatic dissociation as indicated previously (Llamazares-Prada et al 2021). Briefly, the minced tissue (1g) was introduced in GentleMACS C-tubes (Miltenyi Biotec) containing 10 μM ROCK inhibitor (Y-27632, Adooq Bioscience), 10 μg DNAseI (ProSpec-Tany TechnoGene), the enzyme mix from the human tumor tissue dissociation kit (Miltenyi Biotec) and 4.5 mL of CO 2 -independent media (Thermo Fisher Scientific) supplemented with 1% BSA (Carl Roth), 1% penicillin & streptomycin (Fisher Scientific) and 1% Amphotericin B (Fisher Scientific).…”

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics

“…Classical droplet-based scRNA-seq technologies such as Drop-seq, In-Drop, Chromium (10× Genomics, Pleasanton, CA, USA) and Nadia (Dolomite Bio, Royston, UK) are widely used due to their high throughput and reasonable pricing. Although recent approaches in bulk RNA and scRNA-seq applications of cryopreserved cells showed promising results for lung epithelial cells, using viable cells from fresh tissue remains the recommended option by the manufacturers (10× Genomics, Dolomite Bio) of commercially available droplet-based scRNA-seq workflows [ 26 , 27 ]. In plate-based scRNA-seq technologies such as SMART-seq2, single cells are conventionally sorted by flow cytometry into wells in order to overcome the viability shortcoming, as dead cells can be removed during sorting.…”