Authors: Su, Z., Pich-Bavastro, C., & Gaide, O.
53rd European Society for Dermatological Research annual meeting 2024, 04 September 2024.
DOI: 10.1016/j.jid.2024.10.117
Abstract
We have successfully developed a novel state-of-the-art protocol for the large-scale analysis of skin samples by single-cell RNA sequencing (scRNA-seq). We are part of a large research consortium (SEAWave) that aims to provide a comprehensive study of the impact of 5G waves on health. We are leading the work package studying the effects of 5G FR2 (the next-gen 5G frequencies) on human skin, while other groups will perform coordinated studies on cells and animals. In this study, participants (healthy volunteers / patients with dermatoporosis / skin-cancer-prone syndromes / inflamed skin) are exposed to precise doses of 5G, thus generating 168 skin samples, to be analyzed by state-of-the-art scRNA-seq analysis (an unbiased and very sensitive technique, ideal for studying cell behavior changes). A classic scRNA-seq approach would not work well for such a large batch, which requires very high reproducibility. We took advantage of a recently released kit from 10x Genomics (Chromium Next GEM), which enables scRNA-seq analysis of fixed samples. The benefits are to improve reproducibility and flexibility, not to analyze genetic data and to be less biased by mitochondrial and ribosomal genes. However, this protocol had never been used for skin and did not work for this tissue. The main difficulty was obtaining enough intact cells for the scRNA-seq analysis, as skin is known to be quite difficult to dissociate. To obtain the best protocol, we tested fixation at different steps, several enzyme cocktails and incubation times. Our work shows that the best results are obtained by cutting the biopsy in fixation buffer, fixing the samples between 19 and 21 hours, dissociating them in Liberase TL for 1 hour at 37°C and overnight at 4°C, then performing manual dissociation, and finally sorting cells with DAPI staining. Using this refined protocol, we are now able to detect all cell types expected in healthy skin by scRNA-seq analysis. We think that the dermatological community has a lot to gain by using the robust new protocol we have been able to develop.