Skip to main content
SpringerLink
Log in

A Novel Hypothesis and Characterization to Isolate Microvascular Endothelial Cells Simultaneously with Adipose-Derived Stem Cells from the Human Adipose-Derived Stromal Vascular Fraction

  • Original Article
  • Published:
Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Angiogenesis and vasculogenesis are essential processes for successful tissue regeneration in tissue engineering and regenerative medicine. The adipose-derived stromal vascular fraction (SVF) is not only a source of adipose stem cells (ASC) but also a suitable source of microvascular endothelial cells because it is a rich capillary network. So, we propose a new hypothesis for isolating adipose-derived human microvascular endothelial cells (HMVEC-A) from the SVF and developed a dual isolation system that isolates two cell types from one tissue.

Method:

To isolate HMVEC-A, we analyzed the supernatant discarded when ASC is isolated from the adipose-derived SVF. Based on this analysis, we assumed that the SVF adherent to the bottom of the culture plate was divided into two fractions: the stromal fraction as the ASC-rich fraction, and the vascular fraction (VF) as the endothelial cells-rich fraction floating in the culture supernatant. VF isolation was optimized and the efficiency was compared, and the endothelial cells characteristics of HMVEC-A were confirmed by flow cytometric analysis, immunocytochemistry (ICC), a DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) uptake, and in vitro tube formation assay.

Results:

Consistent with the hypothesis, we found a large population of HMVEC-A in the VF and isolated these HMVEC-A by our isolation method. Additionally, this method had higher yields and shorter doubling times than other endothelial cells isolation methods and showed typical morphological and phenotypic characteristics of endothelial cells.

Conclusion:

Cells obtained by the method according to our hypothesis can be applied as a useful source for studies such as tissue-to-tissue networks, angiogenesis and tissue regeneration, patient-specific cell therapy, and organoid chips.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Marrella A, Lee TY, Lee DH, Karuthedom S, Syla D, Chawla A, et al. Engineering vascularized and innervated bone biomaterials for improved skeletal tissue regeneration. Mater Today (Kidlington). 2018;21:362–76.

    Article  CAS  Google Scholar 

  2. Sarker MD, Naghieh S, Sharma NK, Chen X. 3D biofabrication of vascular networks for tissue regeneration: a report on recent advances. J Pharm Anal. 2018;8:277–96.

    Article  CAS  Google Scholar 

  3. Rademakers T, Horvath JM, van Blitterswijk CA, LaPointe VLS. Oxygen and nutrient delivery in tissue engineering: approaches to graft vascularization. J Tissue Eng Regen Med. 2019;13:1815–29.

    Article  CAS  Google Scholar 

  4. Mittal R, Woo FW, Castro CS, Cohen MA, Karanxha J, Mittal J, et al. Organ-on-chip models: implications in drug discovery and clinical applications. J Cell Physiol. 2019;234:8352–80.

    Article  CAS  Google Scholar 

  5. Collins SD, Yuen G, Tu T, Budzinska MA, Spring K, Bryant K, et al. In vitro models of the liver: disease modeling, drug discovery and clinical applications. In: Tirnitz-Parker JEE, editor. Hepatocellular carcinoma. Brisbane (AU): Codon Publications. 2019. Chapter 3.

  6. Olgasi C, Talmon M, Merlin S, Cucci A, Richaud-Patin Y, Ranaldo G, et al. Patient-specific iPSC-derived endothelial cells provide long-term phenotypic correction of hemophilia A. Stem Cell Reports. 2018;11:1391–406.

    Article  CAS  Google Scholar 

  7. Williams IM, Wu JC. Generation of endothelial cells from human pluripotent stem cells. Arterioscler Thromb Vasc Biol. 2019;39:1317–29.

    Article  CAS  Google Scholar 

  8. Sharath SS, Ramu J, Nair SV, Iyer S, Mony U, Rangasamy J. Human adipose tissue derivatives as a potent native biomaterial for tissue regenerative therapies. Tissue Eng Regen Med. 2020;17:123–40.

    Article  CAS  Google Scholar 

  9. Chun SY, Lim JO, Lee EH, Han MH, Ha YS, Lee JN, et al. Preparation and characterization of human adipose tissue-derived extracellular matrix, growth factors, and stem cells: a concise review. Tissue Eng Regen Med. 2019;16:385–93.

    Article  CAS  Google Scholar 

  10. Williams SK, Wang TF, Castrillo R, Jarrell BE. Liposuction-derived human fat used for vascular graft sodding contains endothelial cells and not mesothelial cells as the major cell type. J Vasc Surg. 1994;19:916–23.

    Article  CAS  Google Scholar 

  11. Nishimura S, Manabe I, Nagasaki M, Hosoya Y, Yamashita H, Fujita H, et al. Adipogenesis in obesity requires close interplay between differentiating adipocytes, stromal cells, and blood vessels. Diabetes. 2007;56:1517–26.

    Article  CAS  Google Scholar 

  12. Lee SJ, Lee CR, Kim KJ, Ryu YH, Kim E, Han YN, et al. Optimal condition of isolation from an adipose tissue-derived stromal vascular fraction for the development of automated systems. Tissue Eng Regen Med. 2020;17:203–8.

    Article  CAS  Google Scholar 

  13. Nguyen A, Guo J, Banyard DA, Fadavi D, Toranto JD, Wirth GA, et al. Stromal vascular fraction: a regenerative reality? Part 1: current concepts and review of the literature. J Plast Reconstr Aesthet Surg. 2016;69:170–9.

    Article  Google Scholar 

  14. Varma MJ, Breuls RG, Schouten TE, Jurgens WJ, Bontkes HJ, Schuurhuis GJ, et al. Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev. 2007;16:91–104.

    Article  Google Scholar 

  15. Dykstra JA, Facile T, Patrick RJ, Francis KR, Milanovich S, Weimer JM, et al. Concise review: fat and furious: harnessing the full potential of adipose-derived stromal vascular fraction. Stem Cells Transl Med. 2017;6:1096–108.

    Article  Google Scholar 

  16. Ehrlund A, Acosta JR, Björk C, Hedén P, Douagi I, Arner P, et al. The cell-type specific transcriptome in human adipose tissue and influence of obesity on adipocyte progenitors. Sci Data. 2017;4:170164.

    Article  CAS  Google Scholar 

  17. Ramakrishnan VM, Boyd NL. The adipose stromal vascular fraction as a complex cellular source for tissue engineering applications. Tissue Eng Part B Rev. 2018;24:289–99.

    Article  Google Scholar 

  18. Bi H, Li H, Zhang C, Mao Y, Nie F, Xing Y, et al. Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process. Stem Cell Res Ther. 2019;10:302.

    Article  Google Scholar 

  19. Dobke M, Peterson DR, Mattern RH, Arm DM, Li WW. Microvascular tissue as a platform technology to modify the local microenvironment and influence the healing cascade. Regen Med. 2020;15:1313–28.

    Article  CAS  Google Scholar 

  20. Kim JN, Lee JH, Oh DY, Yoo G, Jeon YJ, Moon SH, et al. Simple, and effective isolation and differentiation of endothelial cells and their progenitor cells from human fat tissue. Tissue Eng Regen Med. 2011;8:A37-43.

  21. Ilesanmi AO. A novel way of harvesting a large population of endothelial cells for clinical and experimental use. Biomed Res Rev. 2018. https://doi.org/10.15761/BRR.1000120.

    Article  Google Scholar 

  22. Schlie S, Gruene M, Dittmar H, Chichkov BN. Dynamics of cell attachment: adhesion time and force. Tissue Eng Part C Methods. 2012;18:688–96.

    Article  CAS  Google Scholar 

  23. Sekiguchi H, Ii M, Jujo K, Yokoyama A, Hagiwara N, Asahara T. Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells. PLoS One. 2011;6:e28639.

    Article  CAS  Google Scholar 

  24. Haynes BA, Huyck RW, James AJ, Carter ME, Gaafar OU, Day M, et al. Isolation, expansion, and adipogenic induction of CD34+CD31+ endothelial cells from human omental and subcutaneous adipose tissue. J Vis Exp. 2018;17:57804.

    Google Scholar 

  25. Van Pham P, Vu NB, Nguyen HT, Phan NK. Isolation of endothelial progenitor cells from human adipose tissue. BMRAT. 2016;3:645–52.

    Google Scholar 

  26. Bora P, Majumdar AS. Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther. 2017;8:145.

    Article  Google Scholar 

  27. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci. 2000;97:3422–7.

    Article  CAS  Google Scholar 

  28. Pellegrinelli V, Carobbio S, Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues. Diabetologia. 2016;59:1075–88.

    Article  CAS  Google Scholar 

  29. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–28.

    Article  CAS  Google Scholar 

  30. Corvera S, Gealekman O. Adipose tissue angiogenesis: impact on obesity and type-2 diabetes. Biochim Biophys Acta. 2014;1842:463–72.

    Article  CAS  Google Scholar 

  31. Fukumura D, Ushiyama A, Duda DG, Xu L, Tam J, Krishna V, et al. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ Res. 2003;93:e88-97.

    Article  CAS  Google Scholar 

  32. Freiman A, Shandalov Y, Rozenfeld D, Shor E, Segal S, Ben-David D, et al. Adipose-derived endothelial and mesenchymal stem cells enhance vascular network formation on three-dimensional constructs in vitro. Stem Cell Res Ther. 2016;7:5.

    Article  Google Scholar 

  33. Gharibi B, Hughes FJ. Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells. Stem Cells Transl Med. 2012;1:771–82.

    Article  CAS  Google Scholar 

  34. Cribbs SK, Martin GS, Rojas M. Monitoring of endothelial dysfunction in critically ill patients: the role of endothelial progenitor cells. Curr Opin Crit Care. 2008;14:354–60.

    Article  Google Scholar 

  35. Pill K, Melke J, Muhleder S, Pultar M, Rohringer S, Priglinger E, et al. Microvascular networks from endothelial cells and mesenchymal stromal cells from adipose tissue and bone marrow: a comparison. Front Bioeng Biotechnol. 2018;6:156.

    Article  Google Scholar 

  36. Pill K, Hofmann S, Redl H, Holnthoner W. Vascularization mediated by mesenchymal stem cells from bone marrow and adipose tissue: a comparison. Cell Regen. 2015;4:8.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2017M3A9E2060428) and by a Ministry of Trade Industry and Energy of Korea (10063334).

Author information

Authors and Affiliations

  1. Department of Biomedicine and Health Sciences, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, Republic of Korea

    Yeon Hee Ryu & Jong-Won Rhie

  2. Department of Plastic and Reconstructive Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, Republic of Korea

    Suk-Ho Moon, Ki Joo Kim, Young Joon Jun, Deuk Young Oh & Jong-Won Rhie

  3. Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea

    Sang‐Heon Kim

Authors
  1. Yeon Hee Ryu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suk-Ho Moon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ki Joo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Young Joon Jun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Deuk Young Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sang‐Heon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jong-Won Rhie
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jong-Won Rhie.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

There are no animal experiments carried out for this article.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ryu, Y.H., Moon, SH., Kim, K.J. et al. A Novel Hypothesis and Characterization to Isolate Microvascular Endothelial Cells Simultaneously with Adipose-Derived Stem Cells from the Human Adipose-Derived Stromal Vascular Fraction. Tissue Eng Regen Med 18, 429–440 (2021). https://doi.org/10.1007/s13770-021-00332-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13770-021-00332-5

Keywords

Search

Navigation