Impact of different tissue dissociation protocols on endothelial cell recovery from developing lungs.. 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.