Antonyshyn Jeremy A, Mazzoli Vienna, McFadden Meghan J, Gramolini Anthony O, Hofer Stefan O P, Simmons Craig A, Santerre Paul J
Institute of Biomedical Engineering, University of Toronto, Toronto, Canada.
Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Toronto, Canada.
Bio Protoc. 2022 May 20;12(10). doi: 10.21769/BioProtoc.4422.
Human adipose tissue-resident microvascular endothelial cells are not only garnering attention for their emergent role in the pathogenesis of obesity-related metabolic disorders, but are also of considerable interest for vascular tissue engineering due, in part, to the abundant, accessible, and uniquely dispensable nature of the tissue. Here, we delineate a protocol for the acquisition of microvascular endothelial cells from human fat. A cheaper, smaller, and simpler alternative to fluorescence-assisted cell sorting for the immunoselection of cells, our protocol adapts magnet-assisted cell sorting for the isolation of endothelial cells from enzymatically digested adipose tissue and the subsequent enrichment of their primary cultures. Strategies are employed to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the reproducible acquisition of human adipose tissue-resident microvascular endothelial cells with purities ≥98%. They exhibit morphological, molecular, and functional hallmarks of endothelium, yet retain a unique proteomic signature when compared with endothelial cells derived from different vascular beds. Their cultures can be expanded for >10 population doublings and can be maintained at confluence for at least 28 days without being overgrown by residual stromal cells from the cell sorting procedure. The isolation of human adipose tissue-resident microvascular endothelial cells can be completed within 6 hours and their enrichment within 2 hours, following approximately 7 days in culture. Graphical abstract.
人脂肪组织驻留微血管内皮细胞不仅因其在肥胖相关代谢紊乱发病机制中的新作用而备受关注，还因其组织丰富、易于获取且具有独特的可舍弃性，在血管组织工程方面具有相当大的研究价值。在此，我们描述了一种从人脂肪中获取微血管内皮细胞的方法。作为荧光辅助细胞分选用于细胞免疫选择的一种更便宜、更小型且更简单的替代方法，我们的方法采用磁辅助细胞分选从酶消化的脂肪组织中分离内皮细胞，并随后富集其原代培养物。我们采用了一些策略来减轻免疫磁性微粒的非特异性摄取，从而能够以≥98%的纯度可重复地获取人脂肪组织驻留微血管内皮细胞。它们表现出内皮细胞的形态、分子和功能特征，但与源自不同血管床的内皮细胞相比，保留了独特的蛋白质组学特征。它们的培养物可以扩增超过10个群体倍增，并且可以在汇合状态下维持至少28天，而不会被细胞分选过程中残留的基质细胞过度生长。人脂肪组织驻留微血管内皮细胞的分离可在6小时内完成，培养约7天后，其富集可在2小时内完成。图形摘要。
