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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
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).
Aghazadeh, Y., Khan, S. T., Nkennor, B. & Nunes, S. S. Cell-based therapies for vascular regeneration: past, present and future. Pharmacol. Ther., 107976 https://doi.org/10.1016/j.pharmthera.2021.107976 (2021).
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).
Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nat. Biotechnol. 23, 879–884 (2005).
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).
Koike, N. et al. Tissue engineering: creation of long-lasting blood vessels. Nature 428, 138–139 (2004).
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).
Sun, X. et al. Transplanted microvessels improve pluripotent stem cell-derived cardiomyocyte engraftment and cardiac function after infarction in rats. Sci. Transl. Med. 12 https://doi.org/10.1126/scitranslmed.aax2992 (2020).
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).
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).
Rodbell, M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239, 375–380 (1964).
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).
Wagner, R. C. & Matthews, M. A. The isolation and culture of capillary endothelium from epididymal fat. Microvasc. Res. 10, 286–297 (1975).
Nunes, S. S., Rekapally, H., Chang, C. C. & Hoying, J. B. Vessel arterial–venous plasticity in adult neovascularization. PLoS ONE 6, e27332 (2011).
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).
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).
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).
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).
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).
Shepherd, B. R. et al. Rapid perfusion and network remodeling in a microvascular construct after implantation. Arterioscl. Thromb. Vasc. Biol. 24, 898–904 (2004).
Krishnan, L. et al. Manipulating the microvasculature and its microenvironment. Crit. Rev. Biomed. Eng. 41, 91–123 (2013).
Nunes, S. S. et al. Implanted microvessels progress through distinct neovascularization phenotypes. Microvasc. Res. 79, 10–20 (2010).
Schulz, T. C. et al. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS ONE 7, e37004 (2012).
Laschke, M. W. et al. Effects of cryopreservation on adipose tissue-derived microvascular fragments. J. Tiss. Eng. Regen. Med. 12, 1020–1030 (2018).
Vunjak-Novakovic, G. et al. Challenges in cardiac tissue engineering. Tiss. Eng. Part B Rev. 16, 169–187 (2010).
Nor, J. E. et al. Engineering and characterization of functional human microvessels in immunodeficient mice. Lab. Invest. 81, 453–463 (2001).
Lesman, A. et al. Transplantation of a tissue-engineered human vascularized cardiac muscle. Tiss. Eng. Part A 16, 115–125 (2010).
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).
Redd, M. A. et al. Patterned human microvascular grafts enable rapid vascularization and increase perfusion in infarcted rat hearts. Nat. Commun. 10, 584 (2019).
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).
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).
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).
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).
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).
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).
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).
Shepherd, B. R., Hoying, J. B. & Williams, S. K. Microvascular transplantation after acute myocardial infarction. Tiss. Eng. 13, 2871–2879 (2007).
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Protocols thanks Juan Melero-Martin, Yun Xia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
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.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-022-00743-1
This article is cited by
-
Generation of Connective Tissue-Free Microvascular Fragment Isolates from Subcutaneous Fat Tissue of Obese Mice
Tissue Engineering and Regenerative Medicine (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
