Abstract
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.
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Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research (PJT 153160 and 180641), the Juvenile Diabetes Research Foundation (3-SRA-2016-251-S-B), the Natural Sciences and Engineering Research Council (RGPIN 06621-2017), an Early Researcher Award from the Ministry of Research, Innovation and Science (ER17 13 420 149), the Stem Cell Network (Horizon Award HZN-C4R1-3) and the University of Toronto’s Medicine by Design initiative, which receives funding from the Canada First Research Excellence Fund. S.S.N. holds the John Kitson McIvor Endowed Chair in Diabetes Research. Y.A. was supported by postdoctoral fellowships from the JDRF-Canadian clinical trial network, Toronto General Hospital Research Institute and Medicine by Design.
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Manuscript writing and editing, X.S., Y.A. and S.S.N. Figure preparation, X.S. and Y.A. All authors contributed to the article and read and approved the submitted version.
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Key references using this protocol
Sun, X. et al. Sci. Transl. Med. 12, eaax2992 (2020): https://doi.org/10.1126/scitranslmed.aax2992
Aghazadeh, Y. et al. Cell Stem Cell 28,1936–1949.e8 (2021): https://doi.org/10.1016/j.stem.2021.08.001
Nunes, S. et al. Microvasc. Res. 79, 10–20 (2010): https://doi.org/10.1016/j.mvr.2009.10.001
Extended data
Extended Data Fig. 1 Optimization of enzymatic treatment.
Digestion time should be experimentally optimized for each lot of collagenase I. We often test approximately five lots at a time, keeping the collagenase concentration constant (2 mg/ml) but varying the incubation time to find the optimal incubation time for a specific lot. a–c, Adipose tissue is harvested (a), minced (b) and subjected to collagenase digestion (c). The enzymatic digestion is monitored over time by sampling it every ~2–3 min for up to ~15 min and observing under the microscope. This will define the optimal incubation time (i.e., the timepoint when MVs are abundant). d–g, Once defined, we repeat that isolation procedure at the optimized timepoint, purify the MVs by sieving (d–f) and then cast MVs in 3D gels at 20,000 MVs/ml of gel (g). h, MV growth is monitored for 1 week. i, Lot selection is based on MV growth in 3D and yield. We select the lot that produces the highest MV yield within the ones that show robust angiogenic growth. Of note, most MVs once cast in 3D collagen gels will grow well; however, yields vary a lot depending on the lot. Once a protocol is established for a new lot, a second experimenter repeats the isolation using the new lot and optimized protocol. The current optimized incubation time in our lab lot is 7.5 min. j, Yields range from 5,000 to 10,000 MVs per milliliter of fat, with three people performing the isolation. Refer to main text for details for a, b and d–g.
Extended Data Fig. 2 Inclusion/exclusion criteria of MVs.
Freshly isolated MVs are counted according to the size and coverage. a,b, MVs can be single tube (a) or bifurcated (b). c, Minimal MV to be included (arrow). Smaller one (double arrowhead) should be excluded. d, Counting also excludes single cells (double arrowhead), debris (arrowhead) and MV with poor coverage (arrow). Scale bar, 200 µm.
Extended Data Fig. 3 pH of collagen hydrogel.
a, Stock collagen is acidic. b, pH of working collagen hydrogel is ~7.4 as verified by pH strips.
Extended Data Fig. 4 Handling collagen constructs.
After the collagen hydrogels have become completely solidified, keep constructs in medium until implantation. a, Tilt the plate to a 45° angle to help hydrogel detach from the bottom of the well. b, Place the forceps around the hydrogels, and pick it up without applying pressure. c, Using a second pair of forceps, gently open the incision made in the dorsal area of the mouse and gently push the hydrogel inside the opening.
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Sun, X., Aghazadeh, Y. & Nunes, S.S. Isolation of ready-made rat microvessels and its applications in effective in vivo vascularization and in angiogenic studies in vitro. Nat Protoc 17, 2721–2738 (2022). https://doi.org/10.1038/s41596-022-00743-1
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DOI: https://doi.org/10.1038/s41596-022-00743-1
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