High-yield isolation and characterization of microvascular niche cells from human full-thickness skin biopsies.

The development of physiologically relevant in vitro vascular models requires access to the cellular components of the microvascular niche. However, most studies rely on a limited subset of vascular or support cells, often using pooled, immortalized, or non-adult sources. To address this limitation, a protocol was developed for the simultaneous isolation of human dermal blood endothelial cells (HDBECs), lymphatic endothelial cells (HDLECs), fibroblasts (HDFs), and pericytes (HDPCs) from adult skin biopsies. Biopsies (25-100 cm²) from donors (age 28-67) were processed using enzymatic and mechanical dissociation, followed by FACS with lineage- and exclusion-specific markers. Purity and identity were verified by flow cytometry and immunofluorescence, and functionality tested in 3D fibrin gels. The protocol yielded all four cell types with high efficiency. Endothelial populations exhibited CD31 and ERG expression, with HDLECs displaying higher PDPN and PROX1 than HDBECs. Support cells showed distinct morphologies, and differences in CD49a and FSP1 enabled discrimination between HDFs and HDPCs. In 3D gels, both endothelial populations formed vascular-like networks with matched support cells. We report a robust method to isolate phenotypically distinct HDBECs, HDLECs, HDFs, and HDPCs from adult skin, supporting the generation of donor-matched microvascular models for aging research, disease modeling, and personalized therapeutic screening.