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.
Anchorage-independent cells possess morphological features and cell membrane compositions that are distinct from adherent cells. They display minimal surface area, have a low rate of endocytosis and generally possess few proteoglycans which make it a challenge to deliver nucleic acids into them. Wide ranges of methods and materials have been developed to tackle the delivery obstacles for the polynucleotide-based therapeutics in modifying non-adherent cells. This article summarizes the techniques and biomaterials that have been utilized for transfection of anchorage-independent cells. First, physical techniques are briefly described along with particular applications for which they are well-suited. The structure-activity relationship of various biomaterial carriers of polynucleotides are then discussed with strategies employed to enhance their capability to transfect anchorage-independent cells. In conclusion, the authors' perspectives on different methods for polynucleotide delivery to primary human cells are compared, along with a discussion of their progression towards clinical trials.
