Impact of different tissue dissociation protocols on endothelial cell recovery from developing lungs.
Developing mouse lungs (day 14 of post-natal life) were dissociated by two different enzymatic methods, one employing commercially available Dispase, and another employing a commercial preparation of purified collagenase. The endothelial cell populations recovered from both approaches were characterized first by flow cytometry, to assess endothelial cell purity and viability. Subsequently, single-cell RNA-Seq was employed to characterize the constituent subpopulations of endothelial cells within each preparation. The single-cell RNA-Seq revealed that endothelial cell populations generated with Dispase exhibited reduced complexity, being enriched largely with one subpopulation of microvascular endothelial cells (gCap2) characterized by H2-Ab1 and Trf expression, whilst endothelial cell populations generated using collagenase yielded a complex endothelial cell population that included endothelial cell sub-types derived from the macro- and microvasculature, including the arterial and venous circulation, as well as the lymphatics. Overall design: Endothelial cells were isolated from mouse lungs on the 14th day of post-natal life. Lungs were cleared by vascular perfused, and enzymatically dissociated using either Dispase of collagenase. Endothelial cells were isolated by from the resultant single-cell suspensions using positive CD31 and negative CD45 selection. Life/dead cell discrimination was undertaken using DAPI exclusion by live cells. Resultant cell preparations were subjected to single-cell RNA-Seq analysis to allow clustering of constituent endothelial cell subtypes by principal component analysis.