Fig. 1: Primary cultures of human adipose tissue-derived microvascular endothelial cells (HAMVECs) are often overgrown by residual adipose tissue-derived stromal/stem cells (ASCs) from the magnet-assisted cell sorting (MACS) procedure.

The stromal vascular fraction of enzymatically digested human subcutaneous abdominal white adipose tissue (a) was depleted of CD45⁺ leucocytes prior to positively selecting for CD31 expression to establish primary cultures of CD45⁻CD31⁺ HAMVECs (b, c). Their primary cultures were often overgrown by residual CD45⁻CD31⁻ ASCs from the MACS procedure despite sequential enrichments for CD31 expression (f, g), prompting the retention of ASCs for downstream studies (d, e). Shown are representative pseudocolour plots depicting the composition of the different populations of cells (a, b, d, f), as well as representative photomicrographs depicting their corresponding morphologies (c, e, g). Scale bars represent 200 µm; values mean ± standard deviation. While the isolation of HAMVECs was initially attempted from twenty patients (n = 20 biologically independent samples), only three cultures were visibly free of stromal cell overgrowth (n = 3 biologically independent samples); in the other 17 patients, cultures were visibly overgrown by stromal cells within 2 weeks (n = 17 biologically independent samples). The composition of the different populations of cells was assessed in three patients in all but visibly contaminated primary cultures of HAMVECs (a, b, d; n = 3 biologically independent samples), in which flow cytometry was used to elucidate the identity of the contaminating stromal cells rather than to assess their purity (f; n = 1 biologically independent sample).