Single cell transcriptome sequencing of fresh and cryopreserved bronchoalveolar lavage cells

<bold>Background/Aim:</bold> Bronchoalveolar lavage (BAL) cells are crucial for lung physiology investigations. Advances in single-cell RNA sequencing (scRNASeq) have enhanced lung research by enabling detailed gene expression analysis at the level of individual cells. This study examines the effects of cryopreservation on the transcriptome of BAL cells compared to fresh samples using the 10x Genomics system. <bold>Methods:</bold> BAL cells from three subjects underwent the scRNAseq protocol, processed either as fresh or cryopreserved using two different freezing media. The Single Cell 3’ Kit and Chromium iX instrument from 10x Genomics were employed. Libraries were sequenced on the NovaSeq 6000. For each 10x run, a minimum of 10,000 cells were loaded. The resulting Fastq files were analyzed using the CellRanger suite and the Seurat (version 5.0.1) Integration package, with GRCh38 serving as the reference genome. <bold>Results:</bold> Cryopreservation led to an increase in mitochondrial gene content, in line with cell viability assessments using Trypan blue staining (with the lowest viability recorded at 78%). Nevertheless, sufficient number of cells remained after filtering for subsequent analysis. The integrated data revealed that cryopreserved and fresh cells displayed similar clustering, with no significant discrepancies in the first 10 principal components. The proportions of CD163- and CD4-positive cells were similar across the conditions. <bold>Conclusion:</bold> Our findings suggest that cryopreservation is a feasible technique for conducting scRNASeq of BAL cells, preserving transcriptomic integrity with minimal discernible alterations.