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Isolation of Microvascular Endothelial Cells
微血管内皮细胞分离   

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本实验方案简略版
Immunity
Nov 2017

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

  1. 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 )

  2. Animals
    Mice (Balb/c, age 6 weeks up to 1 year from Charles River, UK or the in-house breeding facility)

  3. Reagents
    1. Ice
    2. Isoflurane
    3. Phosphate buffered saline solution (PBS, Gibco)
    4. Collagenase type II (Thermo Fisher Scientific, GibcoTM, 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, PharmingenTM, catalog number: 553927 )
      3. Hamster Igs (BD, CompBeadTM, 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 IgG2b 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, PharmingenTM, catalog number: 550274 )
      2. CD105 (Endoglin, clone MJ7/18, rat IgG2a) (BD, PharmingenTM, 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, GibcoTM, catalog number: 41966-052 )
    14. Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030 )
    15. 10,000 U/ml Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    16. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360039 )
    17. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630056 )
    18. 1% non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140050 )
    19. 2-mercaptoethanol (Thermo Fisher Scientific, GibcoTM, 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

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

Procedure

Note: For the EC purification described, tissue from no more than one to two animals is required (see Figure 1 for diagram).


Figure 1. Isolation of endothelial cells (ECs) from tissues by immunomagnetic separation. Tissues consist of diverse cell types and matrix components, and in general, the endothelial content constitutes 1-2% of the total tissue mass. Tissues are mechanically disrupted and enzymatically digested to create a single-cell suspension. Endothelial-specific Abs are added to the single-cell suspension to label the EC. ECs are separated from the other components by using magnetic beads, resulting in highly pure population of ECs.

  1. Euthanize mice according to your local animal regulations. We use an overdose of isoflurane, which leads to breathing arrest within one minute.
  2. Working as sterilely as possible remove murine organs such as heart, lung, and liver using autoclaved instruments and rinse in PBS to remove blood. 
  3. In a Petri dish, using sterile crossed scalpels, dissect tissue into 2-mm3 blocks.
  4. Wash twice in PBS by low-speed centrifugation (210 x g, 1 min).
  5. Incubate diced tissue in a solution of collagenase (0.5 mg/ml) for 1 h at 37 °C in a humid incubator.
    Note: We use Gibco® Collagenase Type II because compared to other collagenase preparations it has a higher clostripain activity and is well-suited for the digestion of heart, bone, thyroid, cartilage, and liver tissues. N.B. it is isolated from Clostridium histolyticum and packaged as a lyophilized, non-sterile powder for research use in cell or tissue dissociation and organ perfusions. Gibco® Collagenase Type II activity is guaranteed to be greater than 125 units/mg.
  6. Subsequently, add 75 µl DNase I solution per 10 ml cell suspension and incubate for another 30 min in a 37 °C water bath with continuous agitation.
  7. Pass digested tissue through a cell strainer to remove undigested blocks.
  8. Rinse the cell strainer twice with PBS supplemented with 2.5% FCS to collect any remaining cell.
  9. Incubate for a further 10 min in 0.25% trypsin (1 ml of trypsin for every 100 mg of tissue) to obtain single cell suspension.
  10. Wash once in 500 µl PBS supplemented with 2.5% FCS.
  11. Incubate for 30 min at 4 °C with murine immunoglobulins to block Fc receptors.
    Note: Mouse BD Fc Block is a purified rat IgG2b anti-mouse CD16/CD32 monoclonal antibody.
  12. Wash twice in cold PBS supplemented with 2.5% FCS.
  13. Incubate for 45 min at 4 °C with rat anti-mouse CD31, rat anti-mouse CD105 and biotinylated isolectin B4.
  14. Wash twice in cold 500 µl PBS supplemented with 0.5% FCS and count cells.
  15. Resuspend pellet and incubate with PBS 0.5% FCS (200 μl/L, 2.5 x 107 cells), rat anti-mouse Ig (25 μl/L, 2.5 x 107 cells)- and streptavidin-conjugated microbeads (25 μl/L, 2.5 x 107 cells) for 15 min at 4 °C (total volume 250 μl). Meanwhile, load columns onto the separation unit (use one column every 1-2.5 x 107 cells) and wash each column with 500 μl PBS 0.5% FCS as per manufacturer’s instructions.
  16. Load each column with 250 μl cell suspension. The magnetically labeled cells are retained in the column(s) while non-labeled cells pass through. After the cell suspension has flowed through the column, wash the column twice with 500 μl PBS 0.5% FCS. For detailed procedure, please refer to the video on the manufacturer's website. 
  17. Unload the column(s) from the magnet and elute the magnetically retained cells with PBS 0.5% FCS using the plunger provided.
  18. Wash the eluted cells and centrifuge at 200 x g for 5 min. Resuspend in EC medium (105 cells/ml) and plate out in dishes/plates (Table 1) pre-coating with gelatin. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage. You can get about 80% of yield.
    Note: Coat P100 dishes with gelatin, let sit in an incubator(37 °C) for at least 30 min, wash in PBS and leave to dry.

    Table 1. Passaging Seeding Density and Volume


  19. After overnight culture on 5% CO2, remove non-adherent cells and replace medium with 75 μg/ml EC growth supplement.
  20. At confluence, detach EC with either trypsin-EDTA or cell dissociation solution and characterize.
    Note: To preserve the features of EC physiology and gene expression, the tissue specimen should preferably be processed immediately after resection. It is advisable to have ready-to-use aliquots of the work solutions in storage.

Data analysis

  1. FACS analysis
    ECs were detached from the culture flasks with trypsin/EDTA (Life Technologies), washed and resuspended in phosphate buffered saline solution (PBS, Gibco) containing 1% FCS (Globepharm). Cells (106 cells/ml) were then incubated with the indicated monoclonal antibody for 30 min at 4 °C. Cells were then washed twice in cold PBS with 1% FCS and incubated for a further 30 min at 4 °C with the appropriate FITC conjugated secondary antibody. After two additional washes, the cells were analyzed using an EPICS Profile Cytometer (Coulter Electronics, Luton, UK).

  2. Immunocytochemistry
    Primary antibody diluted in 3% bovine serum albumin was applied overnight at 4 °C, followed by incubation with a secondary antibody conjugated rhodamine red-X (Molecular Probes) for 1 h at room temperature. Nuclei were labeled with Hoechst 33258 diluted in 3% bovine serum albumin for 15 min at room temperature. All cells were mounted using Dako mounting media (Dako), and fluorescence images were captured using fluorescence microscopy (Zeiss epi-fluorescent microscope).

  3. Results
    1. The surface molecule expression pattern of primary cultures EC express CD31, and CD105 was determined by flow cytometry. As demonstrated, isolated ECs show high-level expression of CD31 and CD105 in both heart and lung after isolation (Figure 2). In addition, murine EC grew in several clusters formed monolayers and demonstrated spindle-shaped and cobblestone-like appearances after ten days as demonstrate under bright field miscopy Figures 3A and 3B. We further investigated the availability of these cells using dye exclusion trypan assay and found that the viability remains high, reaches over 80% after the second passage as shown in Figures 3C and 3D. Figure 3E shows statistical analysis of percentage cell survival in day 2 and day 10 EC seeding.


      Figure 2. Phenotypic characterization of EC purified from murine lung and heart. EC isolates from murine lung (passage 1) were stained with the antibodies specific for the surface molecules indicated within each graph. Isotype-matched irrelevant antibodies were used as a control.


      Figure 3. Bright field images of primary murine lung and heart endothelial cell after isolation. A and B. Isolated ECs were subjected to light microscopy analysis for morphology day 2 (A) and day 7 (B). C, D and E. Trypan blue counting indicates 80% of the cells are surviving on day 2 and 10.

    2. In this protocol, the cultured cells retain their morphological and functional key characteristics of in vivo ECs. We were also able to show the immunofluorescent staining positive cells for CD31 (Figure 4).


      Figure 4. Immunofluorescent characterizations of isolated endothelial cells (ECs). Cells were spun onto microscope slides and subjected to immunofluorescence staining against the indicated antigens. PECAM-1 (MEC 13.3) (green) with 4,6-diamidino-2-phenylindole (DAPI) (blue).

    3. The significance applications of primary cultured mouse endothelial cells
      The protocol provides a great opportunity to study the endothelial-specific activities of targeted molecules. The ability to yield high numbers of mouse ECs makes it very useful in cardiovascular research. This method also reduces cost and improves the potential of studying EC-based therapy in murine models through engraftment of endothelial cells.

  4. Conclusion
    Here we follow this simple and quick method to generate primary EC lines. This can be used for multiple passages for vascular research.

  5. Statistical analysis
    Results are expressed as mean ±SD or SEM, as indicated. The Student’s t-test and ANOVA were used. All reported P values are two-sided. A P-value of less than 0.05 was regarded as significant.

Recipes

  1. EC medium
    Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Paisley, Scotland)
    2 mM glutamine (Gibco)
    100 U/ml penicillin (Gibco)
    100 μg/ml streptomycin (Gibco)
    1 mM sodium pyruvate (Gibco)
    20 mM HEPES (Gibco)
    1% non-essential amino acids (Gibco)
    50 mM 2-mercaptoethanol (Gibco)
    Freshly added 20% heat-inactivated foetal calf serum (FCS; Globepharm, Esher, UK)
    75 μg/ml EC growth supplement (Sigma, Poole, UK)
    Prepared in 2% gelatin (type B from bovine skin, Sigma) coated tissue culture flasks (Nunc, Life Technologies, Paisley, UK)

Acknowledgments

K.CP. Cheung and F.M.Marelli-Berg are supported by the British Heart Foundation grants CH/15/2/32064 and by the Barts Charity grant MGU0377. The authors declare no conflict of interest.

References

  1. Cheung, K., Ma, L., Wang, G., Coe, D., Ferro, R., Falasca, M., Buckley, C. D., Mauro, C. and Marelli-Berg, F. M. (2015). CD31 signals confer immune privilege to the vascular endothelium. Proc Natl Acad Sci U S A 112(43): E5815-5824.
  2. Cid, M. C., Segarra, M., Garcia-Martinez, A. and Hernandez-Rodriguez, J. (2004). Endothelial cells, antineutrophil cytoplasmic antibodies, and cytokines in the pathogenesis of systemic vasculitis. Curr Rheumatol Rep 6(3): 184-194.
  3. Gumkowski, F., Kaminska, G., Kaminski, M., Morrissey, L. W. and Auerbach, R. (1987). Heterogeneity of mouse vascular endothelium. In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells. Blood Vessels 24(1-2): 11-23.
  4. Van Den Berg, T. K., Hasbold, J., Renardel De Lavalette, C., Dopp, E. A., Dijkstra, C. D. and Klaus, G. G. (1996). Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells. Immunology 88(2): 294-300.
  5. Wang, J. M., Huang, Y. J., Wang, Y., Xu, M. G., Wang, L. C., Wang, S. M. and Tao, J. (2007). Increased circulating CD31+/CD42- microparticles are associated with impaired systemic artery elasticity in healthy subjects. Am J Hypertens 20(9): 957-964.

简介

血管内皮是正常血管稳态所必需的。其功能障碍参与各种心血管疾病。小鼠内皮细胞培养是心血管疾病研究的重要工具。该协议演示了一种快速,有效的方法从小鼠组织中分离微血管内皮细胞而无需任何特殊设备。为了分离内皮细胞,将肺或心脏机械切碎并用胶原酶和胰蛋白酶进行酶促消化。然后将获得的单细胞悬浮液与抗CD31,抗CD105抗体和生物素化的异凝集素B-4温育。使用大鼠抗小鼠Ig-和链霉亲和素缀合的微珠,使用磁珠分离收获内皮细胞。扩张内皮细胞并收集用于随后的分析。这些培养物的形态和表型特征在培养10代以上保持稳定。在任何阶段没有过度生长的非内皮来源的污染细胞。

【背景】微血管内皮细胞通过调节白细胞再循环和作为T淋巴细胞的抗原呈递细胞而在免疫应答的发展中起中心作用。内皮的良好状态对血管稳态很重要。功能失调的内皮参与各种心血管疾病,包括动脉粥样硬化,血管炎和缺血/再灌注损伤(Cid et al。,2004; Wang等人,2007)。因此,体外培养的内皮细胞培养物是研究血管生理学和疾病病理学的重要工具。然而,分离原代鼠类内皮细胞被认为是特别困难的,因为大多数描述的方案涉及用消化酶灌注器官或大血管和耗时的纯化过程(Gumkowski等人,1987) 。

该协议的目的是提供一种简单的方法,不需要使用任何特殊设备即可从肺/心脏中分离和扩展内皮细胞。使用这种方法,我们以前补充了体内研究,证明了CD31信号传导在内皮细胞细胞保护中的重要性(Cheung等人,2015年)。

关键字:原代培养, 内皮细胞, 紧密连接, CD31, Pecam1

材料和试剂

  1. 物料
    1. 移液器吸头
    2. 多孔板(细胞培养级)(Greiner Bio One International,目录号:662160)

    3. 50 ml离心管(细胞培养级)(Greiner Bio One International,目录号:210261)
    4. 10毫升一次性吸管(Greiner Bio One International,目录号:607160)
    5. 细胞过滤器(100μm,Corning,目录号:352360; 70μm,Corning,目录号:352350)
    6. 手术刀
    7. miniMACS分离单元(Miltenyi Biotec,目录号:130-042-102)
      注意:标记EC的磁性细胞分选使用包括两个磁体的miniMACS分离单元(Miltenyi Biotec,Bisley,Surrey,UK)进行。将标记的细胞与MACS磁山羊抗 - 大鼠IgG(H + L)(Miltenyi Biotec)MicroBeads和链霉抗生物素蛋白(Miltenyi Biotec)MicroBeads一起温育,然后使用高梯度磁力分离柱(MS > + 栏,Miltenyi Biotec)根据制造商的说明放置在分离单元上。
    8. 高梯度磁力分离柱(MS + 柱)(Miltenyi Biotec,目录号:130-042-201)

  2. 动物
    小鼠(Balb / c,6周龄至英国查尔斯河至1年或内部养殖设施)

  3. 试剂

    1. 异氟醚
    2. 磷酸盐缓冲盐溶液(PBS,Gibco)
    3. II型胶原酶(Thermo Fisher Scientific,Gibco TM,目录号:17101015)
    4. EC媒体
    5. DNaseI解决方案

    6. 0.125%胰蛋白酶在0.2%EDTA(Life Technologies)中
    7. Dako安装媒体(Dako)
    8. 微珠和链霉亲和素(Miltenyi Biotec,目录号:130-048-101)
    9. 抗体
      1. 生物素化的异凝集素B4(购自英国彼得伯勒的Vector Laboratories公司)
        注意:抗CD40 mAb 3/23(大鼠IgG2a)(Van Den Berg等,1996)是G.Klaus博士(英国伦敦国立医学研究院)的一份礼物。
      2. 大鼠IgG2a(克隆R35-95,BD,Pharmingen TM,目录号:553927)
      3. 仓鼠鸡蛋(BD,CompBead TM ,目录号:552845)
      4. 小鼠IgG1(TdT鸡尾酒对照,Harlan Sera-Lab,Oxon,UK,Thermo Fisher Scientific,目录号:31903)
        注意:上述单克隆抗体16b和16c在染色实验中用作同种型匹配的对照抗体:大鼠IgG2a(克隆R35-95);仓鼠Igs。为了阻断Fc受体,使用小鼠IgG2a和小鼠IgG1(TdT鸡尾酒对照,Harlan Sera-Lab,Oxon,UK)。
      5. MACS磁山羊抗大鼠IgG(H + L)(Miltenyi Biotec,目录号:130-048-501)
      6. 大鼠IgG 2b抗小鼠CD16 / CD32单克隆抗体(BD,目录号:553141)
      7. 二抗偶联罗丹明红-X(Molecular Probes)
    10. FACS
      注:以下抗体购自Pharmingen(La Jolla,CA)。
      1. CD31(PECAM-1,克隆MEC13.3,大鼠IgG2a,k)(BD,Pharmingen TM,目录号:550274)
      2. CD105(内皮糖蛋白,克隆MJ7 / 18,大鼠IgG2a)(BD,Pharmingen TM,目录号:550546) 
    11. 免疫荧光染色
      PECAM-1(MEC 13.3)(Santa Cruz Biotechnology,目录号:sc-18916)
    12. Dulbecco改良的Eagle培养基(DMEM,Thermo Fisher Scientific,Gibco TM,目录号:41966-052)
    13. 谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030)
    14. 10,000U / ml青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    15. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360039)
    16. HEPES(Thermo Fisher Scientific,Gibco TM,目录号:15630056)
    17. 1%非必需氨基酸(Thermo Fisher Scientific,Gibco TM,目录号:11140050)
    18. 2-巯基乙醇(Thermo Fisher Scientific,Gibco TM,目录号:31350010)
    19. 热灭活的胎牛血清(FCS; Globepharm,Esher,UK)
    20. EC生长补充剂(Sigma-Aldrich,目录号:E0760)
    21. 2%明胶(来自牛皮的B型,Sigma-Aldrich,目录号:G7765)涂布的组织培养瓶(Nunc,Life Technologies,Paisley,UK)
    22. 工作介质(见食谱)

设备

  1. 移液器
  2. 无菌烧杯100-150毫升(在180°C灭菌)
  3. 层流工作台
  4. 镊子(在180°C灭菌)
  5. 剪刀(180°C灭菌)
  6. 摇床
  7. 水浴
  8. 离心机(Hettich Instruments,型号:UNIVERSAL 320 R)
  9. 固定角转子(海蒂诗仪器,目录号:1620A)
  10. EPICS Profile Cytometer(Coulter Electronics,Luton,UK)
  11. 荧光显微镜(蔡司epi荧光显微镜)

程序

注意:对于所描述的EC纯化,需要不超过一至两只动物的组织(见图1的图)。


图1.通过免疫磁性分离从组织分离内皮细胞(EC)组织由不同的细胞类型和基质组分组成,并且通常,内皮含量占总组织质量的1-2% 。将组织机械破碎并酶促消化以产生单细胞悬液。将内皮特异性抗体加入到单细胞悬液中以标记EC。通过使用磁珠将EC与其他组分分离,导致EC的高纯度群体。

  1. 根据当地的动物法规安乐死小鼠。我们使用过量的异氟醚,导致呼吸停止在一分钟内。
  2. 尽可能无菌操作使用高压灭菌器械清除小鼠器官,如心脏,肺和肝脏,并用PBS冲洗以除去血液。 
  3. 在培养皿中,使用无菌的交叉式解剖刀,将组织切成2mm 3块。
  4. 通过低速离心(210×g,1分钟)在PBS中洗涤两次。
  5. 在潮湿的培养箱中,将胶原酶(0.5 mg / ml)溶液中的切块组织在37°C孵育1小时。
    注:我们使用Gibco II型胶原酶,因为与其他胶原酶制剂相比,它具有更高的梭菌蛋白酶活性,非常适合消化心脏,骨骼,甲状腺,软骨和肝脏组织。注:它是从溶组织梭菌中分离出来的,并且包装成冻干的非无菌粉末,用于研究细胞或组织分离和器官灌流。 Gibco II型胶原酶活性保证大于125单位/ mg。
  6. 随后,每10ml细胞悬液加入75μlDNA酶I溶液,并在连续搅拌下在37℃水浴中再孵育30分钟。
  7. 通过细胞过滤器消化组织消除未消化的块。

  8. 用补充有2.5%FCS的PBS冲洗细胞过滤器两次以收集剩余的细胞。
  9. 在0.25%胰蛋白酶(每100毫克组织含1毫升胰蛋白酶)中继续孵育10分钟以获得单细胞悬液。

  10. 在500μl补充有2.5%FCS的PBS中清洗一次
  11. 在4℃用鼠免疫球蛋白孵育30分钟以阻断Fc受体。
    注意:小鼠BD Fc Block是纯化的大鼠IgG 2b抗小鼠CD16 / CD32单克隆抗体。 br />
  12. 用含2.5%FCS的冷PBS洗两次。
  13. 在4°C与大鼠抗小鼠CD31,大鼠抗小鼠CD105和生物素化异凝集素B4孵育45分钟。
  14. 在冷的500μlPBS中补充0.5%FCS并计数细胞两次。
  15. 重悬沉淀并与PBS 0.5%FCS(200μl/ L,2.5×10 7细胞),大鼠抗小鼠Ig(25μl/ L,2.5×10 7 / sup细胞)和链霉亲和素缀合的微珠(25μl/ L,2.5×10 7细胞)在4℃(总体积250μl)中孵育15分钟。同时,按照制造商的说明将色谱柱装载到分离单元上(每1-2.5 x 10 7个细胞使用一个色谱柱),并用500μlPBS 0.5%FCS清洗每个色谱柱。
  16. 用250μl细胞悬液加载每个色谱柱。磁性标记的细胞保留在柱中,而未标记的细胞通过。细胞悬液流过色谱柱后,用500μlPBS 0.5%FCS洗涤柱子两次。有关详细步骤,请参阅视频在制造商的网站上。 
  17. 从磁铁上卸载柱子,使用提供的柱塞,用PBS 0.5%FCS洗脱磁性保留的细胞。
  18. 洗涤洗脱的细胞并在200×g下离心5分钟。在EC培养基中重悬(10 5细胞/ ml),并在用明胶预涂的碟/盘(表1)中平板化。这些培养物的表型和形态在培养10-15代时保持稳定,并且在任何阶段都未观察到非内皮来源污染细胞的过度生长。你可以获得约80%的收益。
    注意:用明胶包被P100培养皿,放入培养箱(37°C)中至少30分钟,用PBS清洗并晾干。

    表1.传代播种密度和体积


  19. 在5%CO 2中过夜培养后,除去未贴壁细胞并用75μg/ ml EC生长补充剂代替培养基。
  20. 汇合时,将EC与胰蛋白酶-EDTA或细胞解离溶液分开并表征。
    注意:为了保留EC生理和基因表达的特征,组织标本应该在切除后立即进行处理。
    建议在仓储中准备好可用的等分工作解决方案。

数据分析

  1. FACS分析
    将EC用胰蛋白酶/ EDTA(Life Technologies)从培养瓶中分离,洗涤并重悬于含有1%FCS(Globepharm)的磷酸盐缓冲盐水溶液(PBS,Gibco)中。然后将细胞(10 6细胞/ ml)与指定的单克隆抗体在4℃温育30分钟。然后将细胞在含有1%FCS的冷PBS中洗涤两次,并在4℃用适当的FITC缀合的二抗继续孵育30分钟。再次洗涤两次后,使用EPICS Profile Cytometer(Coulter Electronics,Luton,UK)分析细胞。

  2. 免疫细胞化学
    在3%牛血清白蛋白中稀释的一级抗体在4℃下过夜,然后与二级抗体缀合的罗丹明红-X(Molecular Probes)在室温下温育1小时。细胞核在室温下用3%牛血清白蛋白稀释的Hoechst 33258标记15分钟。使用Dako固定介质(Dako)固定所有细胞,并使用荧光显微镜(Zeiss epi荧光显微镜)捕获荧光图像。

  3. 结果
    1. 原代培养的EC表面分子表达模式表达CD31,通过流式细胞术测定CD105。如所证实的,分离后,分离的EC在心脏和肺中显示CD31和CD105的高水平表达(图2)。此外,小鼠EC在几个簇内生长形成单层,并且在明亮的场错误复制图3A和3B下显示出十天后呈现纺锤形和鹅卵石样外观。我们使用染料排斥锥虫试验进一步研究了这些细胞的可用性,发现存活力仍然很高,在第二次传代后达到80%以上,如图3C和3D所示。图3E显示了第2天和第10天EC接种的细胞存活百分比的统计分析。


      图2.从鼠肺和心脏纯化的EC的表型鉴定来自鼠肺(第1代)的EC分离物用每张图中指示的表面分子特异性抗体染色。
      使用同种型匹配的不相关抗体作为对照

      图3.分离后原代鼠肺和心脏内皮细胞的明场图像A和B分离的ECs进行形态学第2天(A)和第7天(B)的光学显微镜分析。 C,D和E.台盼蓝计数显示80%的细胞在第2天和第10天存活。

    2. 在该协议中,培养的细胞保留了体内ECs的形态和功能关键特征。我们也能够显示CD31的免疫荧光染色阳性细胞(图4)。


      图4.分离的内皮细胞(EC)的免疫荧光表征将细胞旋转到显微镜载玻片上并对指定的抗原进行免疫荧光染色。 PECAM-1(MEC 13.3)(绿色)与4,6-二脒基-2-苯基吲哚(DAPI)(蓝色)。

    3. 原代培养的小鼠内皮细胞的显着应用
      该协议提供了一个很好的机会来研究靶向分子的内皮特异性活性。产生大量小鼠EC的能力使其在心血管研究中非常有用。这种方法还降低了成本,并通过植入内皮细胞提高了在小鼠模型中研究基于EC的疗法的潜力。

  4. 结论
    在这里,我们遵循这个简单快捷的方法来生成主EC线。这可以用于血管研究的多个通道。

  5. 统计分析
    如所示,结果表示为平均值±SD或SEM。使用学生的 t - 测试和方差分析。所有报告的P值都是双面的。
    小于0.05的P 值被认为是显着的

食谱

  1. EC介质
    Dulbecco改良的Eagle培养基(DMEM,Gibco BRL,Paisley,苏格兰)
    2mM谷氨酰胺(Gibco)
    100U / ml青霉素(Gibco)
    100μg/ ml链霉素(Gibco)
    1mM丙酮酸钠(Gibco)
    20mM HEPES(Gibco)
    1%非必需氨基酸(Gibco)
    50mM 2-巯基乙醇(Gibco)
    新鲜加入20%热灭活的胎牛血清(FCS; Globepharm,Esher,UK)

    75μg/ ml EC生长补充剂(Sigma,Poole,UK) 用2%明胶(来自牛皮的B型,Sigma)涂覆的组织培养瓶(Nunc,Life Technologies,Paisley,UK)制备

致谢

K.CP. Cheung和F.M.Marelli-Berg得到英国心脏基金会CH / 15/2/32064和Barts Charity授予MGU0377的资助。作者宣称没有利益冲突。

参考

  1. Cheung,K.,Ma,L.,Wang,G.,Coe,D.,Ferro,R.,Falasca,M.,Buckley,C.D。,Mauro,C.and Marelli-Berg,F.M。(2015)。 CD31信号赋予血管内皮免疫特权。 Proc Natl Acad Sci USA 112(43):E5815-5824。
  2. Cid,M.C。,Segarra,M.,Garcia-Martinez,A。和Hernandez-Rodriguez,J。(2004)。 全身性血管炎发病机制中的内皮细胞,抗中性粒细胞胞质抗体和细胞因子 Curr Rheumatol Rep 6(3):184-194。
  3. Gumkowski,F.,Kaminska,G.,Kaminski,M.,Morrissey,L. W.和Auerbach,R.(1987)。 小鼠血管内皮的异质性。 体外研究淋巴管,大血管和微血管内皮细胞。血管 24(1-2):11-23。
  4. Van Den Berg,T. K.,Hasbold,J.,Renardel De Lavalette,C.,Dopp,E.A。,Dijkstra,C.D。和Klaus,G.G。(1996)。 小鼠CD40的特性:树突状细胞和上皮细胞上CD40表位的差异表达。 免疫学 88(2):294-300。
  5. Wang,J.M.,Huang,Y.J.,Wang,Y.,Xu,M.G.,Wang,L.C.,Wang,S.M。和Tao,J。(2007)。 增加循环CD31 + / CD42-微粒与健康受试者的全身动脉弹性受损有关。 Am J Hypertens 20(9):957-964。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
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.
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