Optimization of mouse kidney digestion protocols for single-cell applications.

Single-cell technologies such as flow cytometry and single-cell RNA sequencing (scRNAseq) have allowed for comprehensive characterisation of the kidney cellulome. However, there is disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterising kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: (P1) a scRNAseq protocol; (P2) a multi-tissue digestion kit from Miltenyi Biotec ; and (P3) a protocol established in our laboratory. Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth-muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B-intercalating cells compared to the other techniques. P1 and P3 produced similar yields for most cell types, however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich for specific kidney cell types, may benefit from this comparative analysis.