微血管内皮细胞分离

Kenneth C.P. Cheung; Federica   M.  Marelli-Berg

doi:10.21769/BioProtoc.2886

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Peer-reviewed

Isolation of Microvascular Endothelial Cells

微血管内皮细胞分离

KC Kenneth C.P. Cheung email

FM Federica M. Marelli-Berg

发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2886 浏览次数: 15104

评审: Ivan ZanoniLokesh KalekarSalma Merchant

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The authors used this protocol in:

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Abstract

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine tissues without any special equipment. To isolate endothelial cells, the lung or heart were mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained was then incubated with an anti-CD31, anti-CD105 antibody and with biotinylated isolectin B-4. The endothelial cells were harvested using magnetic bead separation with rat anti-mouse Ig- and streptavidin-conjugated microbeads. Endothelial cells were expanded and collected for subsequent analyses. The morphological and phenotypic features of these cultures remained stable over 10 passages in culture. There was no overgrowth of contaminating cells of non-endothelial origin at any stage.

Keywords: Primary culture (原代培养)

Endothelial cells (内皮细胞)

Tight junctions (紧密连接)

CD31 (CD31)

Pecam1 (Pecam1)

Background

Microvascular endothelial cells play a central role in the development of immune responses by regulating leukocyte recirculation and as antigen presenting cells to T lymphocytes. The wellbeing of the endothelium is essential to vascular homeostasis. The dysfunctional endothelium participates in various cardiovascular disorders, including atherosclerosis, vasculitis and ischemia/reperfusion injuries (Cid et al., 2004; Wang et al., 2007). Therefore, in vitro endothelial cell cultures are important tools for studying vascular physiology and disease pathology. However, the isolation of primary murine endothelial cells is considered particularly difficult because most protocols described have involved the perfusion of organs or large vessels with digesting enzymes and time-consuming purification process (Gumkowski et al., 1987).

The purpose of this protocol is to provide a simple method to isolate and expand endothelial cells from the lung/heart without using any special equipment. Using this method, we previously complemented in vivo studies demonstrating the importance of CD31 signaling in endothelial cells cytoprotection (Cheung et al., 2015).

Materials and Reagents

Materials
1. Pipette tips
2. Multiwell plate (cell culture grade) (Greiner Bio One International, catalog number: 662160 )
3. 50 ml centrifuge tubes (cell culture grade) (Greiner Bio One International, catalog number: 210261 )
4. 10 ml disposable pipette (Greiner Bio One International, catalog number: 607160 )
5. Cell strainers (100 µm, Corning, catalog number: 352360 ; 70 µm, Corning, catalog number: 352350 )
6. Scalpel
7. miniMACS separation unit (Miltenyi Biotec, catalog number: 130-042-102 )
  Note: Magnetic cell sorting of labeled EC was performed using a miniMACS separation unit (Miltenyi Biotec, Bisley, Surrey, UK) including two magnets. Labeled cells were incubated with MACS magnetic goat anti-rat IgG (H+L) (Miltenyi Biotec) MicroBeads and streptavidin (Miltenyi Biotec) MicroBeads and then separated using a high gradient magnetic separation column (MS⁺ columns, Miltenyi Biotec) placed on the separation unit, according to the manufacturer’s instructions.
8. High gradient magnetic separation column (MS⁺ columns) (Miltenyi Biotec, catalog number: 130-042-201 )
Animals
Mice (Balb/c, age 6 weeks up to 1 year from Charles River, UK or the in-house breeding facility)
Reagents
1. Ice
2. Isoflurane
3. Phosphate buffered saline solution (PBS, Gibco)
4. Collagenase type II (Thermo Fisher Scientific, Gibco^TM, catalog number: 17101015 )
5. EC media
6. DNaseI solution
7. 0.125% trypsin in 0.2% EDTA (Life Technologies)
8. Dako mounting media (Dako)
9. MicroBeads and streptavidin (Miltenyi Biotec, catalog number: 130-048-101 )
10. Antibodies
  1. Biotinylated isolectin B4 (purchased from Vector Laboratories, Peterborough, UK)
    Note: The anti-CD40 mAb 3/23 (rat IgG2a) (Van Den Berg et al., 1996) was a kind gift from Dr. G. Klaus (National Institute for Medical Research, London, UK).
  2. Rat IgG2a (clone R35-95, BD, Pharmingen^TM, catalog number: 553927 )
  3. Hamster Igs (BD, CompBead^TM, catalog number: 552845 )
  4. Mouse IgG1 (TdT Cocktail Control, Harlan Sera-Lab, Oxon, UK, Thermo Fisher Scientific, catalog number: 31903 )
    Note: The above mAbs 16b and 16c were used as isotype-matched control antibodies in staining experiments: rat IgG2a (clone R35-95); hamster Igs. To block Fc receptors, mouse IgG2a and mouse IgG1 (TdT Cocktail Control, Harlan Sera-Lab, Oxon, UK) were used.
  5. MACS magnetic goat anti-rat IgG (H+L) (Miltenyi Biotec, catalog number: 130-048-501 )
  6. Rat IgG_2b anti-mouse CD16/CD32 monoclonal antibody (BD, catalog number: 553141 )
  7. Secondary antibody conjugated rhodamine red-X (Molecular Probes)
11. FACS
  Note: The following antibodies were purchased from Pharmingen (La Jolla, CA).
  1. CD31 (PECAM-1, clone MEC 13.3, rat IgG2a, k) (BD, Pharmingen^TM, catalog number: 550274 )
  2. CD105 (Endoglin, clone MJ7/18, rat IgG2a) (BD, Pharmingen^TM, catalog number: 550546 )
12. Immuno Fluorescence Staining
  PECAM-1(MEC 13.3) (Santa Cruz Biotechnology, catalog number: sc-18916 )
13. Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Gibco^TM, catalog number: 41966-052 )
14. Glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25030 )
15. 10,000 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
16. Sodium pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360039 )
17. HEPES (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630056 )
18. 1% non-essential amino acids (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140050 )
19. 2-mercaptoethanol (Thermo Fisher Scientific, Gibco^TM, catalog number: 31350010 )
20. Heat-inactivated fetal calf serum (FCS; Globepharm, Esher, UK)
21. EC growth supplement (Sigma-Aldrich, catalog number: E0760 )
22. 2% gelatin (type B from bovine skin, Sigma-Aldrich, catalog number: G7765 ) coated tissue culture flasks (Nunc, Life Technologies, Paisley, UK)
23. Working medium (see Recipes)

Equipment

Pipettes
Sterile beakers 100-150 ml (sterilize at 180 °C)
Laminar flow work bench
Tweezers (sterilize at 180 °C)
Scissors (sterilize at 180 °C)
Shaker
Water bath
Centrifuge (Hettich Instruments, model: UNIVERSAL 320 R )
Fixed-angle rotor (Hettich Instruments, catalog number: 1620A )
EPICS Profile Cytometer (Coulter Electronics, Luton, UK)
Fluorescence microscopy (Zeiss epi-fluorescent microscope)

Procedure

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如何引用

Cheung, K. C. and Marelli-Berg, F. M. (2018). Isolation of Microvascular Endothelial Cells. Bio-protocol 8(12): e2886. DOI: 10.21769/BioProtoc.2886.