Isolation of ready-made rat microvessels and its applications in effective in vivo vascularization and in angiogenic studies in vitro

• Published in: Nature protocols Vol. 17; no. 12; pp. 2721 - 2738
• Main Authors: Sun, Xuetao, Aghazadeh, Yasaman, Nunes, Sara S.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2022; Nature Publishing Group
• Subjects: 631/1647/1407/651; 631/1647/767/1657; 631/61/2296; Adipose tissue; Adipose Tissue - metabolism; Analytical Chemistry; Angiogenesis; Animals; Biological Techniques; Biomedical and Life Sciences; Blood vessels; Body fat; Cardiomyocytes; Cell culture; Cell Differentiation; Collagen; Collagenase; Computational Biology/Bioinformatics; Engraftment; Growth factors; Hydrogels; In vivo methods and tests; Life Sciences; Lumens; Mice; Microarrays; Microvessels; Neovascularization, Physiologic; Organic Chemistry; Pancreas transplantation; Pancreatic islet transplantation; Pluripotency; Progenitor cells; Protocol; Rats; Regenerative medicine; Stem cell transplantation; Stem cells; Supplements; Tissue engineering; Transplantation; Vascularization
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/s41596-022-00743-1

Despite recent advances in the differentiation of human pluripotent stem cells into multiple cell types for application in replacement therapies, tissue vascularization remains a bottleneck for regenerative medicine. Fragments of primary microvessels (MVs) harvested from adipose tissue retain endothelialized lumens and perivascular cell coverage. We have used these MVs to support the survival and engraftment of transplanted human pluripotent stem cell-derived cardiomyocytes, pancreatic progenitors or primary human islets. MVs connect with host vessels, perfuse with blood and form a hierarchal vascular network in vivo after subcutaneous or intracardiac transplantation. MVs also display the ability to remodel and form stable vascular networks with long-term retention (>3.5 months). MVs can be cultured in 3D hydrogels in vitro, where they retain vessel shape and undergo angiogenic sprouting without the need for exogenous growth factor supplementation. Therefore, MVs offer a robust vascularization strategy for regenerative medicine approaches and a platform for angiogenic studies and drug testing in vitro. Here we describe in detail the protocol for: (1) the isolation of MVs from rat epididymal fat by limited collagenase digestion, followed by size-selective sieving; (2) the incorporation of MVs into 3D collagen hydrogels; (3) the in vitro culture of MVs in 3D gels for angiogenic studies; and (4) the in vivo transplantation of 3D hydrogels containing MVs into the mouse subcutis. The isolation procedure does not require highly specific equipment and can be performed in ~3 h by researchers with experience in rodent handling and cell culture. Protocol for isolating ready-made rat microvessels from adipose tissue and their incorporation into 3D collagen hydrogels. These microvessel gels can be used for in vitro angiogenic assays or transplanted into mice for in vivo vascularization studies.