Background

White adipose tissue is not only the predominant store of energy in the body, but it is also an essential endocrine organ that helps regulate systemic metabolic homeostasis (Stern et al., 2016). While the importance of its resident microvascular endothelial cells for the growth and remodeling of the tissue is well-established, their role in the pathogenesis of obesity-related metabolic disorders is only beginning to be appreciated (Graupera et al., 2018). Our robust protocol for the acquisition of human adipose tissue-derived microvascular endothelial cells (HAMVECs) may facilitate these important investigations at the interface of vascular biology and systemic metabolic homeostasis (Antonyshyn et al., 2021). Moreover, it provides the field of vascular tissue engineering with an abundant, accessible, and uniquely dispensable source of endothelial cells, which may ultimately be used for the vascularization of engineered tissues (Rouwkema et al., 2016) and the endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner (Antonyshyn et al., 2020).

Our protocol employs magnet-assisted cell sorting for the immunoselection of HAMVECs (Antonyshyn et al., 2021). Its low cost, small physical footprint, and ease-of-use make it more amenable for a number of stakeholders when compared with alternatives such as fluorescence-assisted cell sorting. Previous procedures for the acquisition of endothelial cells by magnet-assisted cell sorting have relied on differential adhesion (Hutley et al., 2001), clonal selection (Alphonse et al., 2015), and manual weeding (McGinn et al., 2004) to prevent the overgrowth of their primary cultures by residual stromal cells from the cell sorting procedure. These techniques are labour-intensive, time-consuming, and of uncertain reproducibility. We developed a procedure that does not necessitate these additional techniques to establish temporally stable cultures of HAMVECs (Antonyshyn et al., 2021).

Here, we delineate our straightforward two-staged procedure for the immunomagnetic acquisition of microvascular endothelial cells from human adipose tissue (Antonyshyn et al., 2021). The first step involves the isolation of the endothelial cells from enzymatically digested fat, and the second step involves the enrichment of their primary cultures to eliminate residual adipose tissue-derived stromal/stem cells (ASCs) from the cell sorting procedure. The stromal vascular fraction of enzymatically digested fat is depleted of CD45⁺ leukocytes prior to positively selecting for CD45^–CD31⁺ HAMVECs. This sequential immunomagnetic selection is used to address challenges arising from the high prevalence of leukocytes in adipose tissue and their capacity to express characteristic endothelial markers, including CD31. The magnet-assisted cell sorting procedure employs immunomagnetic microparticles of a specific size and binding moiety, specifically comprising monodispersed superparamagnetic spheres of a 4.8 µm modal diameter conjugated with antibodies via cleavable deoxyribonucleic acid (DNA) linkers. This defined size distribution of immunomagnetic microparticles prevents their non-specific uptake by ASCs during the labeling portion of the cell sorting procedure, and the cleavable DNA linkers enable the exclusion of the superparamagnetic spheres from primary cultures where they can be more readily internalized by any residual ASCs. These are strategies that mitigate the non-specific uptake of immunomagnetic microparticles by ASCs—which is imperative for the effective isolation and enrichment of endothelial cells by magnet-assisted cell sorting. This facile two-staged procedure enables the reproducible acquisition of HAMVECs with purities ≥98%, which is sufficient to prevent their overgrowth by ASCs and facilitate their use for downstream investigations and regenerative medicine applications. Moreover, the CD45^–CD31^– ASCs may also be retained for downstream investigations (Antonyshyn et al., 2019), having been previously validated to meet the phenotypic criteria delineated by the International Federation for Adipose Therapeutics and Science and the International Society for Cellular Therapy (Bourin et al., 2013).