Isolation and Culture of Mouse Placental Endothelial Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1752)

Abstract

Isolation and culture of endothelial cells (ECs) is a useful tool to study the cellular processes involved in vascular development and vascular maturation. In this chapter, we describe a method to isolate and culture endothelial cells from placentae. This method takes advantage of two transgenes: ROSA26 mT/mG , which drives the expression of GFP upon Cre-mediated recombination, and Tie2-Cre, which expresses Cre driven by the Tie2 promoter in endothelial progenitors and their descendants. GFP-expressing endothelial cells are isolated through fluorescence-activated cell sorting (FACS). The sorted cells express the endothelial marker CD31. This method can be used to study the morphological and physiological properties of placental endothelial cells in mice carrying mutations affecting vascular development.

Key words

Mouse placenta Endothelial cells Fluorescence-activated cell sorting Primary cell culture 

Notes

Acknowledgments

We thank The SickKids-UHN Flow Cytometry Facility for help with FACS, and The Centre for Phenogenomics (TCP) for mouse husbandry. This work was supported by the Heart and Stroke Foundation of Canada (G-17-0018613), the Natural Sciences and Engineering Research Council of Canada (NSERC) (500865), the Canadian Institutes of Health Research (CIHR) (PJT-149046), and Operational Funds from the Hospital for Sick Children to P.D.-O.

References

  1. 1.
    Larrivee B, Karsan A (2005) Isolation and culture of primary endothelial cells. Methods Mol Biol 290:315–329PubMedGoogle Scholar
  2. 2.
    Nachman RL, Jaffe EA (2004) Endothelial cell culture: beginnings of modern vascular biology. J Clin Invest 114(8):1037–1040. https://doi.org/10.1172/JCI23284 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gulino-Debrac D (2013) Mechanotransduction at the basis of endothelial barrier function. Tissue Barriers 1(2):e24180. https://doi.org/10.4161/tisb.24180 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Benelli R, Albini A (1999) In vitro models of angiogenesis: the use of Matrigel. Int J Biol Markers 14(4):243–246CrossRefPubMedGoogle Scholar
  5. 5.
    Cheung K, Ma L, Wang G, Coe D, Ferro R, Falasca M, Buckley CD, Mauro C, Marelli-Berg FM (2015) CD31 signals confer immune privilege to the vascular endothelium. Proc Natl Acad Sci U S A 112(43):E5815–E5824. https://doi.org/10.1073/pnas.1509627112 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dong QG, Bernasconi S, Lostaglio S, De Calmanovici RW, Martin-Padura I, Breviario F, Garlanda C, Ramponi S, Mantovani A, Vecchi A (1997) A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler Thromb Vasc Biol 17(8):1599–1604CrossRefPubMedGoogle Scholar
  7. 7.
    Marelli-Berg FM, Peek E, Lidington EA, Stauss HJ, Lechler RI (2000) Isolation of endothelial cells from murine tissue. J Immunol Methods 244(1–2):205–215CrossRefPubMedGoogle Scholar
  8. 8.
    Jin GZ, Park JH, Lee EJ, Wall IB, Kim HW (2014) Utilizing PCL microcarriers for high-purity isolation of primary endothelial cells for tissue engineering. Tissue Eng Part C Methods 20(9):761–768. https://doi.org/10.1089/ten.TEC.2013.0348 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605. https://doi.org/10.1002/dvg.20335 CrossRefPubMedGoogle Scholar
  10. 10.
    Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230(2):230–242. https://doi.org/10.1006/dbio.2000.0106 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Translational MedicineThe Hospital for Sick Children, Peter Gilgan Centre for Research and LearningTorontoCanada
  2. 2.Translational MedicineThe Hospital for Sick ChildrenTorontoCanada
  3. 3.Department of Molecular GeneticsUniversity of TorontoTorontoCanada
  4. 4.Heart & Stroke/Richard Lewar Centres of Excellence in Cardiovascular ResearchTorontoCanada

Personalised recommendations