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


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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Fig. 1: Overview of MV isolation from rat epidydimal fat.
Fig. 2: Step-by-step visualization of the fat MV isolation procedure.
Fig. 3: 3D culture of MVs in collagen hydrogel.
Fig. 4: Neovascular progression in subcutaneously implanted MV grafts.
Fig. 5: Stepwise imaging of MV after isolation, embedding in hydrogels and transplantation in vivo.
Fig. 6: Ready-made MVs get perfused early and persist long term, integrating into the cardiac vasculature and forming stable grafts.

Data availability

The data presented in ‘Anticipated results’ were previously published and are available in the original publications8,9,22.


  1. Sun, X., Altalhi, W. & Nunes, S. S. Vascularization strategies of engineered tissues and their application in cardiac regeneration. Adv. Drug Deliv. Rev. 96, 183–194 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Aghazadeh, Y., Khan, S. T., Nkennor, B. & Nunes, S. S. Cell-based therapies for vascular regeneration: past, present and future. Pharmacol. Ther., 107976 (2021).

  3. Tracy, E. P. et al. State of the field: cellular and exosomal therapeutic approaches in vascular regeneration. Am. J. Physiol. Heart Circ. Physiol. 322, H647–H680 (2022).

    Article  CAS  PubMed  Google Scholar 

  4. Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 23, 879–884 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, Z. Z. et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nat. Biotechnol. 25, 317–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Koike, N. et al. Tissue engineering: creation of long-lasting blood vessels. Nature 428, 138–139 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J. & Langer, R. Endothelial cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA 99, 4391–4396 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sun, X. et al. Transplanted microvessels improve pluripotent stem cell-derived cardiomyocyte engraftment and cardiac function after infarction in rats. Sci. Transl. Med. 12 (2020).

  9. Aghazadeh, Y. et al. Microvessels support engraftment and functionality of human islets and hESC-derived pancreatic progenitors in diabetes models. Cell Stem Cell 28, 1936–1949 e1938 (2021).

    Article  CAS  PubMed  Google Scholar 

  10. Hoying, J. B., Boswell, C. A. & Williams, S. K. Angiogenic potential of microvessel fragments established in three-dimensional collagen gels. Vitr. Cell. Dev. Biol. Anim. 32, 409–419 (1996).

    Article  CAS  Google Scholar 

  11. Rodbell, M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239, 375–380 (1964).

    Article  CAS  PubMed  Google Scholar 

  12. Wagner, R. C., Kreiner, P., Barrnett, R. J. & Bitensky, M. W. Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc. Natl Acad. Sci. USA 69, 3175–3179 (1972).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wagner, R. C. & Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286–297 (1975).

    Article  CAS  PubMed  Google Scholar 

  14. Nunes, S. S., Rekapally, H., Chang, C. C. & Hoying, J. B. Vessel arterial–venous plasticity in adult neovascularization. PLoS ONE 6, e27332 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Laschke, M. W. et al. Vascularisation of porous scaffolds is improved by incorporation of adipose tissue-derived microvascular fragments. Eur. Cells Mater. 24, 266–277 (2012).

    Article  CAS  Google Scholar 

  16. Altalhi, W., Hatkar, R., Hoying, J. B., Aghazadeh, Y. & Nunes, S. S. Type I diabetes delays perfusion and engraftment of 3D constructs by impinging on angiogenesis; which can be rescued by hepatocyte growth factor supplementation. Cell. Mol. Bioeng. 12, 443–454 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Altalhi, W., Sun, X., Sivak, J. M., Husain, M. & Nunes, S. S. Diabetes impairs arterio-venous specification in engineered vascular tissues in a perivascular cell recruitment-dependent manner. Biomaterials 119, 23–32 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Rhoads, R. P. et al. Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway. Am. J. Physiol. Cell Physiol. 296, C1321–1328 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rhoads, R. P. et al. Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro. Biochem. Biophys. Res. Commun. 440, 399–404 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Shepherd, B. R. et al. Rapid perfusion and network remodeling in a microvascular construct after implantation. Arterioscl. Thromb. Vasc. Biol. 24, 898–904 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Krishnan, L. et al. Manipulating the microvasculature and its microenvironment. Crit. Rev. Biomed. Eng. 41, 91–123 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Nunes, S. S. et al. Implanted microvessels progress through distinct neovascularization phenotypes. Microvasc. Res. 79, 10–20 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Schulz, T. C. et al. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS ONE 7, e37004 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Laschke, M. W. et al. Effects of cryopreservation on adipose tissue-derived microvascular fragments. J. Tiss. Eng. Regen. Med. 12, 1020–1030 (2018).

    Article  CAS  Google Scholar 

  25. Vunjak-Novakovic, G. et al. Challenges in cardiac tissue engineering. Tiss. Eng. Part B Rev. 16, 169–187 (2010).

    Article  Google Scholar 

  26. Nor, J. E. et al. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab. Invest. 81, 453–463 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Lesman, A. et al. Transplantation of a tissue-engineered human vascularized cardiac muscle. Tiss. Eng. Part A 16, 115–125 (2010).

    Article  CAS  Google Scholar 

  28. Stevens, K. R. et al. Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue. Proc. Natl Acad. Sci. USA 106, 16568–16573 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Redd, M. A. et al. Patterned human microvascular grafts enable rapid vascularization and increase perfusion in infarcted rat hearts. Nat. Commun. 10, 584 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gerbin, K. A., Yang, X., Murry, C. E. & Coulombe, K. L. Enhanced electrical integration of engineered human myocardium via intramyocardial versus epicardial delivery in infarcted rat hearts. PLoS ONE 10, e0131446 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Shadrin, I. Y. et al. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nat. Commun. 8, 1825 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Nunes, S. S. et al. Angiogenic potential of microvessel fragments is independent of the tissue of origin and can be influenced by the cellular composition of the implants. Microcirculation 17, 557–567 (2010).

    PubMed  PubMed Central  Google Scholar 

  33. Laschke, M. W. et al. Adipose tissue-derived microvascular fragments from aged donors exhibit an impaired vascularisation capacity. Eur. Cells Mater. 28, 287–298 (2014).

    Article  CAS  Google Scholar 

  34. Laschke, M. W. et al. High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments. Eur. Cells Mater. 38, 188–200 (2019).

    Article  CAS  Google Scholar 

  35. Spater, T. et al. Adipose tissue-derived microvascular fragments from male and female fat donors exhibit a comparable vascularization capacity. Front. Bioeng. Biotechnol. 9, 777687 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Honek, J. et al. Modulation of age-related insulin sensitivity by VEGF-dependent vascular plasticity in adipose tissues. Proc. Natl Acad. Sci. USA 111, 14906–14911 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shepherd, B. R., Hoying, J. B. & Williams, S. K. Microvascular transplantation after acute myocardial infarction. Tiss. Eng. 13, 2871–2879 (2007).

    Article  Google Scholar 

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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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Correspondence to Sara S. Nunes.

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Nature Protocols thanks Juan Melero-Martin, Yun Xia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Sun, X. et al. Sci. Transl. Med. 12, eaax2992 (2020):

Aghazadeh, Y. et al. Cell Stem Cell 28,1936–1949.e8 (2021):

Nunes, S. et al. Microvasc. Res. 79, 10–20 (2010):

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. ac, 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). dg, Once defined, we repeat that isolation procedure at the optimized timepoint, purify the MVs by sieving (df) 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 dg.

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).

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